Combined Treatment with an EGFR Kinase Inhibitor and an Agent that Sensitizes Tumor Cells to the Effects of EGFR Kinase Inhibitors

ABSTRACT

The present invention provides a method for treating tumors or tumor metastases in a patient, comprising administering to said patient simultaneously or sequentially a therapeutically effective amount of a combination of an EGFR kinase inhibitor and an agent that sensitizes tumor cells to the effects of EGFR kinase inhibitors, wherein said agent is an mTOR inhibitor that binds to and directly inhibits both mTORC1 and mTORC2 kinases. The present invention also provides a pharmaceutical composition comprising an EGFR kinase inhibitor and an mTOR inhibitor that binds to and directly inhibits both mTORC1 and mTORC2 kinases, in a pharmaceutically acceptable carrier. A preferred example of an EGFR kinase inhibitor that can be used in practicing the methods of this invention is the compound erlotinib HCl (also known as TARCEVA®).

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application which claims priority toU.S. application Ser. No. 12/631,116, which is a divisional of U.S.application Ser. No. 11/717,545, filed Mar. 13, 2007, now U.S. Pat. No.7,651,687, which claims the benefit of U.S. Provisional Application No.60/781,877 filed Mar. 13, 2006, all of which are herein incorporated byreference in their entirety.

BACKGROUND OF THE INVENTION

The present invention is directed to compositions and methods fortreating cancer patients. Cancer is a generic name for a wide range ofcellular malignancies characterized by unregulated growth, lack ofdifferentiation, and the ability to invade local tissues andmetastasize. These neoplastic malignancies affect, with various degreesof prevalence, every tissue and organ in the body.

A multitude of therapeutic agents have been developed over the past fewdecades for the treatment of various types of cancer. The most commonlyused types of anticancer agents include: DNA-alkylating agents (e.g.,cyclophosphamide, ifosfamide), antimetabolites (e.g., methotrexate, afolate antagonist, and 5-fluorouracil, a pyrimidine antagonist),microtubule disrupters (e.g., vincristine, vinblastine, paclitaxel), DNAintercalators (e.g., doxorubicin, daunomycin, cisplatin), and hormonetherapy (e.g., tamoxifen, flutamide). More recently, gene targetedtherapies, such as protein-tyrosine kinase inhibitors (e.g. imatinib;the EGFR kinase inhibitor, erlotinib) have increasingly been used incancer therapy.

The epidermal growth factor receptor (EGFR) family comprises fourclosely related receptors (HER1/EGFR, HER2, HER3 and HER4) involved incellular responses such as differentiation and proliferation.Over-expression of the EGFR kinase, or its ligand TGF-alpha, isfrequently associated with many cancers, including breast, lung,colorectal, ovarian, renal cell, bladder, head and neck cancers,glioblastomas, and astrocytomas, and is believed to contribute to themalignant growth of these tumors. A specific deletion-mutation in theEGFR gene (EGFRvIII) has also been found to increase cellulartumorigenicity. Activation of EGFR stimulated signaling pathways promotemultiple processes that are potentially cancer-promoting, e.g.proliferation, angiogenesis, cell motility and invasion, decreasedapoptosis and induction of drug resistance. Increased HER1/EGFRexpression is frequently linked to advanced disease, metastases and poorprognosis. For example, in NSCLC and gastric cancer, increased HER1/EGFRexpression has been shown to correlate with a high metastatic rate, poortumor differentiation and increased tumor proliferation.

Mutations which activate the receptor's intrinsic protein tyrosinekinase activity and/or increase downstream signaling have been observedin NSCLC and glioblastoma. However the role of mutations as a principlemechanism in conferring sensitivity to EGF receptor inhibitors, forexample erlotinib (TARCEVA®) or gefitinib (IRESSA™), has beencontroversial. Recently, a mutant form of the full length EGF receptorhas been reported to predict responsiveness to the EGF receptor tyrosinekinase inhibitor gefitinib (Paez, J. G. et al. (2004) Science304:1497-1500; Lynch, T. J. et al. (2004) N. Engl. J. Med.350:2129-2139). Cell culture studies have shown that cell lines whichexpress the mutant form of the EGF receptor (i.e. H3255) were moresensitive to growth inhibition by the EGF receptor tyrosine kinaseinhibitor gefitinib, and that much higher concentrations of gefitinibwas required to inhibit the tumor cell lines expressing wild type EGFreceptor. These observations suggests that specific mutant forms of theEGF receptor may reflect a greater sensitivity to EGF receptorinhibitors, but do not identify a completely non-responsive phenotype.

The development for use as anti-tumor agents of compounds that directlyinhibit the kinase activity of the EGFR, as well as antibodies thatreduce EGFR kinase activity by blocking EGFR activation, are areas ofintense research effort (de Bono J. S. and Rowinsky, E. K. (2002) Trendsin Mol. Medicine 8:S19-S26; Dancey, J. and Sausville, E. A. (2003)Nature Rev. Drug Discovery 2:92-313). Several studies have demonstrated,disclosed, or suggested that some EGFR kinase inhibitors might improvetumor cell or neoplasia killing when used in combination with certainother anti-cancer or chemotherapeutic agents or treatments (e.g. Herbst,R. S. et al. (2001) Expert Opin. Biol. Ther. 1:719-732; Solomon, B. etal (2003) Int. J. Radiat. Oncol. Biol. Phys. 55:713-723; Krishnan, S. etal. (2003) Frontiers in Bioscience 8, e1-13; Grunwald, V. and Hidalgo,M. (2003) J. Nat. Cancer Inst. 95:851-867; Seymour L. (2003) CurrentOpin. Investig. Drugs 4(6):658-666; Khalil, M. Y. et al. (2003) ExpertRev. Anticancer Ther. 3:367-380; Bulgaru, A. M. et al. (2003) ExpertRev. Anticancer Ther. 3:269-279; Dancey, J. and Sausville, E. A. (2003)Nature Rev. Drug Discovery 2:92-313; Ciardiello, F. et al. (2000) Clin.Cancer Res. 6:2053-2063; and Patent Publication No: US 2003/0157104).

Erlotinib (e.g. erlotinib HCl, also known as TARCEVA® or OSI-774) is anorally available inhibitor of EGFR kinase. In vitro, erlotinib hasdemonstrated substantial inhibitory activity against EGFR kinase in anumber of human tumor cell lines, including colorectal and breast cancer(Moyer J. D. et al. (1997) Cancer Res. 57:4838), and preclinicalevaluation has demonstrated activity against a number of EGFR-expressinghuman tumor xenografts (Pollack, V. A. et al (1999) J. Pharmacol. Exp.Ther. 291:739). More recently, erlotinib has demonstrated promisingactivity in phase I and II trials in a number of indications, includinghead and neck cancer (Soulieres, D., et al. (2004) J. Clin. Oncol.22:77), NSCLC (Perez-Soler R, et al. (2001) Proc. Am. Soc. Clin. Oncol.20:310a, abstract 1235), CRC (Oza, M., et al. (2003) Proc. Am. Soc.Clin. Oncol. 22:196a, abstract 785) and MBC (Winer, E., et al. (2002)Breast Cancer Res. Treat. 76:5115a, abstract 445). In a phase III trial,erlotinib monotherapy significantly prolonged survival, delayed diseaseprogression and delayed worsening of lung cancer-related symptoms inpatients with advanced, treatment-refractory NSCLC (Shepherd, F. et al.(2005) N. Engl. J. Med. 353(2):123-132). While most of the clinicaltrial data for erlotinib relate to its use in NSCLC, preliminary resultsfrom phase I/II studies have demonstrated promising activity forerlotinib and capecitabine/erlotinib combination therapy in patientswith wide range of human solid tumor types, including CRC (Oza, M., etal. (2003) Proc. Am. Soc. Clin. Oncol. 22:196a, abstract 785) and MBC(Jones, R. J., et al. (2003) Proc. Am. Soc. Clin. Oncol. 22:45a,abstract 180). In November 2004 the U.S. Food and Drug Administration(FDA) approved TARCEVA® for the treatment of patients with locallyadvanced or metastatic non-small cell lung cancer (NSCLC) after failureof at least one prior chemotherapy regimen. TARCEVA® is the only drug inthe epidermal growth factor receptor (EGFR) class to demonstrate in aPhase III clinical trial an increase in survival in advanced NSCLCpatients.

An anti-neoplastic drug would ideally kill cancer cells selectively,with a wide therapeutic index relative to its toxicity towardsnon-malignant cells. It would also retain its efficacy against malignantcells, even after prolonged exposure to the drug. Unfortunately, none ofthe current chemotherapies possess such an ideal profile. Instead, mostpossess very narrow therapeutic indexes. Furthermore, cancerous cellsexposed to slightly sub-lethal concentrations of a chemotherapeuticagent will very often develop resistance to such an agent, and quiteoften cross-resistance to several other antineoplastic agents as well.Additionally, for any given cancer type one frequently cannot predictwhich patient is likely to respond to a particular treatment, even withnewer gene-targeted therapies, such as EGFR kinase inhibitors, thusnecessitating considerable trial and error, often at considerable riskand discomfort to the patient, in order to find the most effectivetherapy.

Thus, there is a need for more efficacious treatment for neoplasia andother proliferative disorders, and for more effective means fordetermining which tumors will respond to which treatment. Strategies forenhancing the therapeutic efficacy of existing drugs have involvedchanges in the schedule for their administration, and also their use incombination with other anticancer or biochemical modulating agents.Combination therapy is well known as a method that can result in greaterefficacy and diminished side effects relative to the use of thetherapeutically relevant dose of each agent alone. In some cases, theefficacy of the drug combination is additive (the efficacy of thecombination is approximately equal to the sum of the effects of eachdrug alone), but in other cases the effect is synergistic (the efficacyof the combination is greater than the sum of the effects of each druggiven alone).

Target-specific therapeutic approaches, such as erlotinib, are generallyassociated with reduced toxicity compared with conventional cytotoxicagents, and therefore lend themselves to use in combination regimens.Promising results have been observed in phase I/II studies of erlotinibin combination with bevacizumab (Mininberg, E. D., et al. (2003) Proc.Am. Soc. Clin. Oncol. 22:627a, abstract 2521) and gemcitabine(Dragovich, T., (2003) Proc. Am. Soc. Clin. Oncol. 22:223a, abstract895). Recent data in NSCLC phase III trials have shown that first-lineerlotinib or gefitinib in combination with standard chemotherapy did notimprove survival (Gatzemeier, U., (2004) Proc. Am. Soc. Clin. Oncol.23:617 (Abstract 7010); Herbst, R. S., (2004) Proc. Am. Soc. Clin.Oncol. 23:617 (Abstract 7011); Giaccone, G., et al. (2004) J. Clin.Oncol. 22:777; Herbst, R., et al. (2004) J. Clin. Oncol. 22:785).However, pancreatic cancer phase III trials have shown that first-lineerlotinib in combination with gemcitabine did improve survival (OSIPharmaceuticals/Genentech/Roche Pharmaceuticals Press Release, Sep. 20,2004).

Activation of EGFR triggers multiple cascades of signal transductionpathways. EGFR contains at least six autophosphorylation sites thatserve as docking nodes for a multitude of intracellular signalingmolecules including adapter proteins and other enzymes. Therefore,rather than regulating a single linear pathway, activation of EGFRmodulates entire networks of cellular signal transduction cascades.These signals affect both cell cycle progression/proliferation andapoptosis. Two signal transduction cascades that lie downstream of EGFRare the MAPK (mitogen activated protein kinase) and Akt pathways. In theMAPK pathway, EGFR activates the small GTP binding protein Ras totransfer cell growth signals through the Raf-MEK-ERK cascade,culminating in the regulation of transcription factors important forcell cycle progression.

EGFR can activate PI3K (through homodimers or heterodimers with HER3) toinitiate signals through the PDK1-Akt pathway. Akt can positivelyregulate anti-apoptotic factors within the cell to promote cellsurvival. In addition Akt can activate the protein kinase mTOR(mammalian target of rapamycin) to promote cell growth andproliferation. mTOR is a major regulator of cell growth andproliferation in response to both growth factors and cellular nutrients.It is a key regulator of the rate limiting step for translation of mRNAinto protein, the binding of the ribosome to mRNA. Here mTOR directlymodulates the activities of a number of downstream signaling proteinsinvolved in protein synthesis. Two substrates that are directlyphosphorylated by mTOR include 4EBP1 and p70S6K. 4EBP1 is atranscriptional repressor that binds to eIF4E, blocking properorganization of the ribosome initiation complex. Phosphorylation of4EBP1 by mTOR disrupts interactions with eIF4E, liberating eIF4E fortranslation. mTOR also directly phosphorylates and activates p70S6K,which in turn phosphorylates S6 ribosomal protein, leading to enhancedmRNA translation.

mTOR exists in at least 2 distinct multiprotein complexes described asraptor-mTOR complex (mTORC1) and rictor-mTOR complex (mTORC2) inmammalian cells (sometimes referred to as just TORC1 and TORC2). mTORC1is composed of mTOR, GβL and raptor proteins and it binds toFKBP12-rapamycin. mTORC1 is a rapamycin-sensitive complex as its kinaseactivity is inhibited by FKB12-rapamycin in vitro. How FKBP12-rapamycininhibits mTOR kinase activity is poorly understood. The drug rapamycindoes not displace GβL or raptor from mTOR but does strongly destabilizethe raptor-mTOR interaction. Extensive work with rapamycin indicatesthat mTORC1 complex positively regulates cell growth. The raptor branchof the mTOR pathway modulates number of processes, including mRNAtranslation, ribosome biogenesis, nutrient metabolism and autophagy. Thetwo mammalian proteins, S6 Kinase 1 (S6K1) and 4E-BP1, which are linkedto protein synthesis, are downstream targets of mTORC1. mTORC1 has beenshown to phosphorylates S6K1 at T389 and is inhibited byFKBP12-rapamycin in vitro and by rapamycin in vivo. mTORC1 can alsophosphorylate 4E-BP1 at T37/46 in vitro and in vivo.

mTORC2 is composed of mTOR, GβL and rictor proteins and it does not bindto FKBP12-rapamycin complex. mTORC2 is a rapamycin-insensitive complexas its kinase activity is not inhibited by FKBP12-rapamycin complex invitro. It is unclear why FKBP12-rapamycin complex does not bind therictor containing mTORC2 complex. Rictor or an unidentified component ofthe complex may block or occupy the FKBP12-rapamycin complex bindingsite or allosterically destroy the FKBP12-rapamycin complex bindingpocket. It has been discovered recently that mTORC2 is a hydrophobicmotif kinase for Akt/PKB and plays an important role in Akt/PKBactivation. mTORC2 has been shown to phosphorylate PKB/Akt at S473 invitro and in vivo. Akt/PKB is a key component of insulin/PI3K signalingpathway and modulates cell survival and proliferation through downstreamsubstrates such as the FOXO class of transcription factors and p53regulator mdm2. In addition, mTORC2 regulates the actin cytoskeletonthrough unknown mechanisms that involve PKCa and Rho. mTORC2 can alsophosphorylate 4E-BP1 in vitro and in vivo.

Deregulation of mTOR pathway is emerging as a common theme in diversehuman diseases and as a consequence drugs that target mTOR havetherapeutic values. The diseases most clearly associated withderegulation of mTORC1 are tuberous sclerosis complex (TSC) andLymphangioleiomyomatosis (LAM), both of which are cause by mutations inTSC1 or TSC2 tumor suppressors. Patients with TSC develop benign tumorsthat when present in brain, however, can cause seizures, mentalretardation and death. LAM is a serious lung disease. Inhibition ofmTORC1 may help patients with Peutz-Jeghers cancer-prone syndrome causedby LKB1 mutation.

mTORC1 may also have role in the genesis of sporadic cancers.Inactivation of several tumor suppressors, in particular PTEN, p53, VHLand NF1, has been linked to mTORC1 activation. Rapamycin and itsanalogues (eg CCI-779, RAD001 and AP23573) inhibit TORC1 and have shownmoderate anti-cancer activity in phase II clinical trials. However, dueto the negative signal from S6K1 to the insulin/PI3K/Akt pathway, it isimportant to note that inhibitors of mTORC1, like rapalogs, can activatePKB/Akt. If this effect persists with chronic rapamycin treatment it mayprovide cancer cells with an increased survival signal that may beclinically undesirable. The PI3K/Akt pathway is activated in manycancers. Activated Akt regulates cell survival, cell proliferation andmetabolism by phosphorylating proteins such as BAD, FOXO, NF-ηB,p21^(Cip1), p27^(Kip1), GSK3β and others. AKT might also promote cellgrowth by phosphorylating TSC2. AKT activation probably promotescellular transformation and resistance to apoptosis by collectivelypromoting growth, proliferation and survival, while inhibiting apoptoticpathways. An inhibitor of both mTORC1 and mTORC2 should be beneficialfor treatment of tumors with elevated AKT phosphorylation, and shoulddown-regulate cell growth, cell survival and cell proliferation.

Recent reports have shown that the sensitivity of cell lines to growthinhibition by EGFR inhibitors is dependent on the down-regulation of thePI3K-Akt pathway. There can be extensive overlap in signaling where anEGFR signaling pathway can also be regulated by several other receptortyrosine kinases. This potential for multiple inputs in EGFR signalingpathways suggests that inhibiting EGFR alone may not allow for growthinhibition of all tumor cells and highlights the potential formulti-point intervention utilizing combinations of receptor tyrosinekinase inhibitors. Combining EGFR inhibitors with inhibitors of IGF1-Rhas shown success in some preclinical models. In addition to multipleinputs in growth factor signaling, specific mutations or proteindeletions in downstream signaling pathways can affect sensitivity toEGFR inhibition. For example the MDA-468 breast tumor cell line containsa deletion of PTEN, and endogenous inhibitor of PI3K signaling.Reconstitution of PTEN in these cells enhances their sensitivity to EGFRinhibition. Such studies have suggested that combining EGFR inhibitorswith agents, such as mTOR inhibitors, that antagonize downstreamsignaling pathways may permit enhanced sensitization in cell lines thateither have redundancy in receptor tyrosine kinase signaling or containspecific mutations in downstream signaling.

Many inhibitors of mTOR have been identified and several are in clinicaltrials for the treatment of cancer (e.g. RAD001 (also known asEverolimus; Novartis); CCI-779 (also known as Temsirolimus; Wyeth);AP23573 (Ariad Pharmaceuticals); and KU-0059475 (Kudus Pharmaceuticals);Mita, M. M. et al. (2003) Cancer Biology & Therapy 2:4:Suppl. 1,S169-S177). The potential effectiveness of combinations of such mTORinhibitors with other anti-cancer agents has also been suggested and isbeing tested in clinical trials (Adjei, A. and Hidalgo, M. (2005) J.Clin. Oncol. 23:5386-5403). Such combinations include combinations ofmTOR inhibitors with protein-tyrosine kinase inhibitors (Sawyers, C.(2003) Cancer Cell 4:343-348; Gemmill, R. M. et al. (2005) Br. J. Cancer92(12):2266-2277; Goudar, R. K. et al. (2005) Mol. Cancer Therapeutics4(1):101-112; International Patent Publication WO 2004/004644; Birle, D.C., et al. Proc. Am. Assoc. Cancer Res. (2nd edn) (2003) 44: 932 Abs.R4692).

Despite the advances in treatment described above there remains acritical need for improved treatments for many human cancers. Theinvention described herein provides new anti-cancer combinationtherapies that are an improvement on the efficacy of either EGFR kinaseinhibitors or mTOR inhibitors when administered alone. In particular,the present invention is directed to methods of combined treatment ofbreast, colon, NSCL or pancreatic cancer with an epidermal growth factorreceptor (EGFR) kinase inhibitor and an mTOR inhibitor that sensitizestumor cells of these cancers to the effects of EGFR kinase inhibitors, aresult which has not previously been reported in the medical literature.

SUMMARY OF THE INVENTION

The present invention provides a method for treating NSCL, pancreatic,colon or breast cancer tumors or tumor metastases in a patient,comprising administering to the patient simultaneously or sequentially atherapeutically effective amount of a combination of an EGFR kinaseinhibitor and an agent that sensitizes tumor cells to the effects ofEGFR kinase inhibitors, wherein said agent is an mTOR inhibitor, with orwithout additional agents or treatments, such as other anti-cancer drugsor radiation therapy.

The present invention also provides a method for treating tumors ortumor metastases in a patient, comprising administering to said patientsimultaneously or sequentially a therapeutically effective amount of acombination of an EGFR kinase inhibitor and an agent that sensitizestumor cells to the effects of EGFR kinase inhibitors, wherein said agentis an mTOR inhibitor that binds to and directly inhibits both mTORC1 andmTORC2 kinases.

The present invention also provides a pharmaceutical compositioncomprising an EGFR kinase inhibitor and an mTOR inhibitor that binds toand directly inhibits both mTORC1 and mTORC2 kinases, in apharmaceutically acceptable carrier.

A preferred example of an EGFR kinase inhibitor that can be used in thisinvention is the compound erlotinib HCl (also known as TARCEVA®).

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Sensitivity of 22 cell lines derived from four tumor types togrowth inhibition by erlotinib. Data are expressed as maximal cellgrowth at 72 hours in the presence of 10 μM erlotinib as compared tocells treated with DMSO alone. A 50% inhibition in maximal cell growthwas used as a cutoff criteria for distinguishing sensitive fromrelatively insensitive cell lines.

FIG. 2: Immunoblot showing the effect of erlotinib on pEGFR, pERK, andpS6 in a panel of 3 sensitive and 3 relatively insensitive cell lines.Cells were treated with erlotinib or DMSO vehicle alone for 2 hours,lysates were prepared and run on gels for western blot.

FIG. 3: A. Sensitivity of cell lines to rapamycin. Data are expressed asmaximal cell growth at 72 hours in the presence of 30 nM rapamycin ascompared to cells treated with DMSO alone. B. Sensitivity of cell linesto the combination of erlotinib and rapamycin. Synergy, as noted by apositive Bliss value, was observed in 13 of 22 cell lines. Thecalculation of Bliss values are described in the materials and methodssection.

FIG. 4: Immunoblot showing that rapamycin down-regulates pS6 (235/236)in cell lines are either sensitive or relatively insensitive toerlotinib. Cells were treated with rapamycin, erlotinib, or thecombination for 2 hours. Where indicated 2 ng/ml EGF ligand was addedfor 8 minutes prior to cell lysis.

FIG. 5: Effect of varying concentrations of rapamycin on theproliferation of three sensitive cell lines (H358, H292, and BxPC3) andthree relatively insensitive cell lines (H460, Calu6, and HCT-116) inthe presence or absence of erlotinib. The dashed line represents theBliss additivity curve and represents the theoretical expectation if thecombined effects of erlotinib with rapamycin were exactly additive.Results shown are a representative of three or more independentexperiments.

FIG. 6: Effect of a combination of erlotinib with rapamycin tosynergistically inhibit growth in a Calu6 xenograft model. In the leftpanel the synergistic effect of the combination on tumor volume ismonitored over a period of 19 days. In the right panel the data for day19 are presented separately.

FIG. 7: The proliferation of both epithelial and mesenchymal NSCLC andpancreatic cells, and ovarian and head and neck squamous cell carcinomacells, are sensitive to the mTOR inhibitor Compound A as a single agent.Sensitivity of 23 cell lines derived from four tumor types to growthinhibition by Compound A. Data are expressed as maximal cell growth at72 hours in the presence of 20 μM Compound A as compared to cellstreated with DMSO alone. A 50% inhibition in maximal cell growth may beused as a cutoff criteria for distinguishing sensitive from relativelyinsensitive cell lines.

FIG. 8: The combination of the mTOR inhibitor Compound A and the EGFRkinase inhibitor erlotinib is synergistic in mesenchymal NSCLC andpancreatic tumor cells. The effect of the combination of compound A anderlotinib in a panel of non-small cell lung cancer (NSCLC) andpancreatic cancer cell lines. The table summarizes for each cell line:the tumor type from which the cell line was derived, EMT status denotesthe expression of epithelial or mesenchymal protein markers. Thecombination is described as additive or synergistic as assessed by thebliss additivity model. the EC50 for Compound A, the maximal growthinhibition of cells cultured in the presence of 20 μM compound A for 72hours, expressed as a percent of cells treated with DMSO alone, the EC50for the combination of compound A+10 μM erlotinib, and the maximalgrowth inhibition at 72 hours for cells cultured in the presence of 20μM Compound A+10 μM erlotinib.

FIG. 9: The combination of the mTOR inhibitor Compound A and the EGFRkinase inhibitor erlotinib is synergistic in multiple tumor cell types.The effect of the combination of compound A and erlotinib in a panel ofovarian cancer, head and neck squamous cell carcinoma(HNSCC) and breastcancer cell lines. The table summarizes for each cell line: the tumortype from which the cell line was derived, EMT status denotes theexpression of epithelial or mesenchymal protein markers. The combinationis described as additive or synergistic as assessed by the blissadditivity model. the EC50 for Compound A, the maximal growth inhibitionof cells cultured in the presence of 20 μM compound A for 72 hours,expressed as a percent of cells treated with DMSO alone, the EC50 forthe combination of compound A+10 μM erlotinib, and the maximal growthinhibition at 72 hours for cells cultured in the presence of 20 μMCompound A+10 μM erlotinib.

FIG. 10: The combination of the EGFR kinase inhibitor erlotinib and themTOR inhibitor Compound A is synergistic in mesenchymal NSCLC cells andadditive in epithelial NSCLC cells. Effect of varying concentrations ofCompound A on the proliferation of (A) four mesenchymal NSCL cell lines(H1703, Calu6, SW1573 and H460) and (B) four epithelial NSCL cell lines(H322, H441, H358 and H292) in the presence or absence of erlotinib. Thedotted line denotes the Bliss additivity curve and represents thetheoretical expectation if the combined effects of erlotinib withCompound A were exactly additive. Results shown are a representative ofthree or more independent experiments.

FIG. 11: The combination of erlotinib and the mTOR inhibitor Compound Ais additive or synergistic in pancreatic cancer cells. Effect of varyingconcentrations of Compound A on the proliferation of three pancreaticcell lines (BxPC3, A1165 and MiaPaCa2) in the presence or absence oferlotinib. The dotted line denotes the Bliss additivity curve andrepresents the theoretical expectation if the combined effects oferlotinib with Compound A were exactly additive. Results shown are arepresentative of three or more independent experiments.

FIG. 12: The combination of the mTOR inhibitor Compound A and the EGFRkinase inhibitor erlotinib is synergistic in multiple tumor cell types.Effect of varying concentrations of Compound A on the proliferation ofrepresentative ovarian (Igrov1, CA-OV-3 and Ovcar-3) and HNSCC (1483)cell lines in the presence or absence of erlotinib. The dotted linedenotes the Bliss additivity curve and represents the theoreticalexpectation if the combined effects of erlotinib with Compound A wereexactly additive. Results shown are a representative of three or moreindependent experiments.

FIG. 13: The combination of the mTOR inhibitor Compound A and the EGFRkinase inhibitor erlotinib enhances apoptosis in pancreatic cancer andNSCLC cells. Effect of the combination of erlotinib with compound A oninduction of apoptosis in NSCL and pancreatic cell lines. For each cellline, fold induction in caspase 3/7 activity relative to DMSO control(white bars) is shown for cells treated with 20 μM Compound A (lightgray bars), 10 μM erlotinib (dark gray bars) and the combination of 20μM Compound A plus 10 μM erlotinib (black bars). Error bars representthe standard deviation between a minimum of two replicates. Data isrepresentative of 3 independent experiments. EMT status for each cellline is denoted as epithelial (E) or mesenchymal (M). The tumor typefrom which each cell line was derived is shown.

FIG. 14: The combination of the mTOR inhibitor Compound A and the EGFRkinase inhibitor erlotinib enhances apoptosis in ovarian cancer cells.Effect of the combination of erlotinib with compound A on induction ofapoptosis in ovarian cell lines. For each cell line, fold induction incaspase 3/7 activity relative to DMSO control (white bars) is shown forcells treated with 20 μM Compound A (light gray bars), 10 μM erlotinib(dark gray bars) and the combination of 20 μM Compound A plus 10 μMerlotinib (black bars). Error bars represent the standard deviationbetween a minimum of two replicates. Data is representative of 2independent experiments.

FIG. 15: The mTOR inhibitor Compound B has single agent activity inovarian cancer and HNSCC cells. Sensitivity of 16 cell lines derivedfrom two tumor types (ovarian, white bars, HNSCC, gray bars) to growthinhibition by Compound B. Data are expressed as maximal cell growth at72 hours in the presence of 10 μM Compound B as compared to cellstreated with DMSO alone. A 50% inhibition in maximal cell growth wasused as a cutoff criteria for distinguishing sensitive from relativelyinsensitive cell lines.

FIG. 16: The combination of the mTOR inhibitor Compound B and the EGFRkinase inhibitor erlotinib is synergistic in multiple tumor cell types.Effect of varying concentrations of Compound B on the proliferation ofrepresentative HNSCC (MSK922 and 1186) and ovarian (SKOV-3 and Igrov1)cell lines in the presence or absence of erlotinib. The dotted linedenotes the Bliss additivity curve and represents the theoreticalexpectation if the combined effects of erlotinib with Compound B wereexactly additive. Results shown are a representative of three or moreindependent experiments.

FIG. 17: The combination of the EGFR kinase inhibitor erlotinib and themTOR inhibitor Compound B enhances apoptosis in ovarian cancer cells.Effect of the combination of erlotinib with compound B on induction ofapoptosis in a representative ovarian and HNSCC cell line. For each cellline, fold induction in caspase 3/7 activity relative to DMSO control(white bars) is shown for cells treated with 20 μM Compound A (lightgray bars), 10 μM erlotinib (dark gray bars) and the combination of 20μM Compound A plus 10 μM erlotinib (black bars). Error bars representthe standard deviation between a minimum of two replicates. Data isrepresentative of 2 independent experiments.

DETAILED DESCRIPTION OF THE INVENTION

The term “cancer” in an animal refers to the presence of cellspossessing characteristics typical of cancer-causing cells, such asuncontrolled proliferation, immortality, metastatic potential, rapidgrowth and proliferation rate, and certain characteristic morphologicalfeatures. Often, cancer cells will be in the form of a tumor, but suchcells may exist alone within an animal, or may circulate in the bloodstream as independent cells, such as leukemic cells.

“Cell growth”, as used herein, for example in the context of “tumor cellgrowth”, unless otherwise indicated, is used as commonly used inoncology, where the term is principally associated with growth in cellnumbers, which occurs by means of cell reproduction (i.e. proliferation)when the rate the latter is greater than the rate of cell death (e.g. byapoptosis or necrosis), to produce an increase in the size of apopulation of cells, although a small component of that growth may incertain circumstances be due also to an increase in cell size orcytoplasmic volume of individual cells. An agent that inhibits cellgrowth can thus do so by either inhibiting proliferation or stimulatingcell death, or both, such that the equilibrium between these twoopposing processes is altered.

“Tumor growth” or “tumor metastases growth”, as used herein, unlessotherwise indicated, is used as commonly used in oncology, where theterm is principally associated with an increased mass or volume of thetumor or tumor metastases, primarily as a result of tumor cell growth.

“Abnormal cell growth”, as used herein, unless otherwise indicated,refers to cell growth that is independent of normal regulatorymechanisms (e.g., loss of contact inhibition). This includes theabnormal growth of: (1) tumor cells (tumors) that proliferate byexpressing a mutated tyrosine kinase or over-expression of a receptortyrosine kinase; (2) benign and malignant cells of other proliferativediseases in which aberrant tyrosine kinase activation occurs; (4) anytumors that proliferate by receptor tyrosine kinases; (5) any tumorsthat proliferate by aberrant serine/threonine kinase activation; and (6)benign and malignant cells of other proliferative diseases in whichaberrant serine/threonine kinase activation occurs.

The term “treating” as used herein, unless otherwise indicated, meansreversing, alleviating, inhibiting the progress of, or preventing,either partially or completely, the growth of tumors, tumor metastases,or other cancer-causing or neoplastic cells in a patient. The term“treatment” as used herein, unless otherwise indicated, refers to theact of treating.

The phrase “a method of treating” or its equivalent, when applied to,for example, cancer refers to a procedure or course of action that isdesigned to reduce or eliminate the number of cancer cells in an animal,or to alleviate the symptoms of a cancer. “A method of treating” canceror another proliferative disorder does not necessarily mean that thecancer cells or other disorder will, in fact, be eliminated, that thenumber of cells or disorder will, in fact, be reduced, or that thesymptoms of a cancer or other disorder will, in fact, be alleviated.Often, a method of treating cancer will be performed even with a lowlikelihood of success, but which, given the medical history andestimated survival expectancy of an animal, is nevertheless deemed anoverall beneficial course of action.

The term “an agent that sensitizes tumor cells to the effects of EGFRkinase inhibitors” when used herein without further qualification as tothe nature of the agent, refers to an mTOR inhibitor.

The phrase “mTOR inhibitor that binds to and directly inhibits bothmTORC1 and mTORC2 kinases” when used herein refers to an mTOR inhibitorthat interacts with and reduces the kinase activity of both mTORC1 andmTORC2 complexes.

The term “therapeutically effective agent” means a composition that willelicit the biological or medical response of a tissue, system, animal orhuman that is being sought by the researcher, veterinarian, medicaldoctor or other clinician.

The term “therapeutically effective amount” or “effective amount” meansthe amount of the subject compound or combination that will elicit thebiological or medical response of a tissue, system, animal or human thatis being sought by the researcher, veterinarian, medical doctor or otherclinician.

The present invention derives from research that provided methods fordetermining which tumors will respond most effectively to treatment withEGFR kinase inhibitors (Thompson, S. et al. (2005) Cancer Res.65(20):9455-9462) based on whether the tumor cells have undergone anepithelial to mesenchymal transition (“EMT”; Thiery, J. P. (2002) Nat.Rev. Cancer 2:442-454; Savagner, P. (2001) Bioessays 23:912-923; Kang Y.and Massague, J. (2004) Cell 118:277-279; Julien-Grille, S., et al.Cancer Research 63:2172-2178; Bates, R. C. et al. (2003) Current Biology13:1721-1727; Lu Z., et al. (2003) Cancer Cell. 4(6):499-515). This workdemonstrated that epithelial cells respond well to EGFR kinaseinhibitors, but that after an EMT the cells become much less sensitiveto such inhibitors. Such knowledge of the cellular characteristicsassociated with sensitivity to EGFR kinase inhibitors, and a knowledgeof the biochemical pathways that regulate EMT, or the reverse process, amesenchymal-to-epithelial transition (MET), allows one to design agents,such as the mTOR inhibitors described herein, that sensitize tumor cellsto the effects of EGFR kinase inhibitors, enabling relativelyinsensitive cells to become sensitive, or sensitive cells to haveincreased sensitivity. Biomarkers can be used to determine whether tumorcells have undergone an EMT (Thomson, S. et al. (2005) Cancer Res.65(20):9455-9462).

The data presented in the Examples herein below demonstrate that mTORinhibitors are agents that can sensitize NSCL, pancreatic, colon orbreast cancer tumor cells to the effects of EGFR kinase inhibitors. Thusthe anti-tumor effects of a combination of an EGFR kinase inhibitor andsuch an agent are superior to the anti-tumor effects of either inhibitorby itself, and co-administration of an mTOR inhibitor with an EGFRkinase inhibitor can be effective for treatment of patients withadvanced cancers such as NSCL, pancreatic, colon or breast cancers. Thesensitizing effect of mTOR inhibitors is seen most frequently in tumorcells that have undergone an EMT, or are relatively insensitive to EGFRkinase inhibitors. In such cells, synergy is frequently observed when anEGFR kinase inhibitor and mTOR inhibitor are used in combination toinhibit tumor cell growth.

Accordingly, the present invention provides a method for treating NSCL,pancreatic, colon or breast cancer tumors or tumor metastases in apatient, comprising administering to said patient simultaneously orsequentially a therapeutically effective amount of a combination of anEGFR kinase inhibitor and an mTOR inhibitor. The present invention alsoprovides a method for treating NSCL, pancreatic, colon or breast cancertumors or tumor metastases in a patient, comprising administering tosaid patient simultaneously or sequentially a synergistically effectivetherapeutic amount of a combination of an EGFR kinase inhibitor and anmTOR inhibitor. The present invention also provides a method fortreating NSCL, pancreatic, colon or breast cancer tumors or tumormetastases in a patient, comprising administering to said patientsimultaneously or sequentially a therapeutically effective amount of acombination of an EGFR kinase inhibitor and an agent that sensitizestumor cells to the effects of EGFR kinase inhibitors, wherein said agentis an mTOR inhibitor. In an embodiment of any of the above methods, thecells of the NSCL, pancreatic, colon or breast cancer tumors or tumormetastases have high sensitivity or are very sensitive to growthinhibition by EGFR kinase inhibitors such as erlotinib as single agents(i.e. without any agent that sensitizes the tumor cells to the effectsof EGFR kinase inhibitors), such as epithelial cells that have notundergone any form of EMT (e.g. H292, H358, or BxPC3 tumor cells). Inanother embodiment of any of the above methods, the cells of the NSCL,pancreatic, colon or breast cancer tumors or tumor metastases have lowsensitivity or are relatively insensitive or refractory to growthinhibition by EGFR kinase inhibitors such as erlotinib as single agents,such as epithelial cells that have undergone an EMT and have acquiredmesenchymal characteristics (e.g. H460 or Calu6 tumor cells).

In a further embodiment of the above methods, the patient to be treatedis tested prior to treatment using a diagnostic assay to determine thesensitivity of tumor cells to an EGFR kinase inhibitor. Any method knownin the art that can determine the sensitivity of the tumor cells of apatient to an EGFR kinase inhibitor can be employed. For example, amethod to determine a patient's likely responsiveness to an EGFR kinaseinhibitor can comprise assessing whether the tumor cells have undergonean epithelial-mesenchymal transition (EMT), by for example determiningthe expression level of one or more tumor cell epithelial and/ormesenchymal biomarkers, thus identifying the patient as one who is lesslikely or not likely to demonstrate an effective response to treatmentwith an EGFR kinase inhibitor as a single agent if their tumor cellshave undergone an EMT (e.g. see Thompson, S. et al. (2005) Cancer Res.65(20):9455-9462 and US Published Patent Application US-2006-0211060-A1,both incorporated herein by reference). For example, the expressionlevel of one or more tumor cell epithelial biomarkers E-cadherin, Brk,γ-catenin, α1-catenin, α2-catenin, α-catenin, keratin 8, keratin 18,connexin 31, plakophilin 3, stratifin 1, laminin alpha-5, or ST14 can beassessed, a high level indicating that the tumor cells have probably notundergone an EMT. Similarly, the expression level of one or more tumorcell mesenchymal biomarkers vimentin, fibronectin 1, fibrillin-1,fibrillin-2, collagen alpha2(IV), collagen alpha2(V), LOXL1, nidogen,C11orf9, tenascin, N-cadherin, tubulin alpha-3, or epimorphin can beassessed, a high level indicating that the tumor cells have probablyundergone an EMT. Other methods that may be utilized to assess thesensitivity of the tumor cells of a patient to an EGFR kinase inhibitorinclude determining the presence of mutant forms of EGFR known to conferan enhanced sensitivity to EGFR kinase inhibitors, or directlydetermining in a tumor cell biopsy the sensitivity of a patients tumorcells to an EGFR kinase inhibitor.

In the above embodiments where the patient is tested prior to treatmentusing a diagnostic assay to determine the sensitivity of tumor cells toan EGFR kinase inhibitor, in one embodiment, when the patient isidentified as one whose tumor cells are predicted to have lowsensitivity to an EGFR kinase inhibitor as a single agent, and thusbased on the results described herein, are likely to display enhancedsensitivity in the presence of an mTOR inhibitor, the patient isadministered, simultaneously or sequentially, a therapeuticallyeffective amount of a combination of an EGFR kinase inhibitor and anmTOR inhibitor. In another embodiment, when the patient is identified asone whose tumor cells are predicted to have high sensitivity to an EGFRkinase inhibitor as a single agent, but may also display enhancedsensitivity in the presence of an mTOR inhibitor based on the resultsdescribed herein, the patient is administered, simultaneously orsequentially, a therapeutically effective amount of a combination of anEGFR kinase inhibitor and an mTOR inhibitor. For these methods, anexample of a preferred EGFR kinase inhibitor would be erlotinib,including pharmacologically acceptable salts or polymorphs thereof. Inthese methods one or more additional anti-cancer agents or treatmentscan be co-administered simultaneously or sequentially with the EGFRkinase inhibitor and mTOR inhibitor, as judged to be appropriate by theadministering physician given the prediction of the likelyresponsiveness of the patient to the combination of EGFR kinaseinhibitor and mTOR inhibitor, in combination with any additionalcircumstances pertaining to the individual patient.

Accordingly, the present invention provides a method for treating NSCL,pancreatic, colon or breast cancer tumors or tumor metastases in apatient, comprising the steps of diagnosing a patient's likelyresponsiveness to an EGFR kinase inhibitor, and administering to saidpatient simultaneously or sequentially a therapeutically effectiveamount of a combination of an EGFR kinase inhibitor and an mTORinhibitor.

The present invention also provides a method for treating NSCL,pancreatic, colon or breast cancer tumors or tumor metastases in apatient, comprising the steps of diagnosing a patient's likelyresponsiveness to an EGFR kinase inhibitor, identifying the patient asone whose tumor or tumor metastases cells are relatively insensitive toan EGFR kinase inhibitor as a single agent, and thus likely to show anenhanced response in the presence of an mTOR inhibitor, andadministering to said patient simultaneously or sequentially atherapeutically effective amount of a combination of an EGFR kinaseinhibitor and an mTOR inhibitor.

The present invention also provides a method for treating NSCL,pancreatic, colon or breast cancer tumors or tumor metastases in apatient, comprising the steps of diagnosing a patient's likelyresponsiveness to an EGFR kinase inhibitor, identifying the patient asone whose tumor or tumor metastases cells are relatively sensitive to anEGFR kinase inhibitor as a single agent, and may thus show an enhancedresponse in the presence of an mTOR inhibitor, and administering to saidpatient simultaneously or sequentially a therapeutically effectiveamount of a combination of an EGFR kinase inhibitor and an mTORinhibitor.

The present invention also provides a method for treating NSCL,pancreatic, colon or breast cancer tumors or tumor metastases in apatient, comprising the steps of diagnosing a patient's likelyresponsiveness to an EGFR kinase inhibitor by assessing whether thetumor cells have undergone an epithelial-mesenchymal transition, andadministering to said patient simultaneously or sequentially atherapeutically effective amount of a combination of an EGFR kinaseinhibitor and an mTOR inhibitor.

The present invention also provides a method for treating NSCL,pancreatic, colon or breast cancer tumors or tumor metastases in apatient, comprising the steps of diagnosing a patient's likelyresponsiveness to an EGFR kinase inhibitor by assessing whether thetumor cells have undergone an epithelial-mesenchymal transition,identifying the patient as one whose tumor or tumor metastases cellshave undergone an epithelial-mesenchymal transition and are thuspredicted to be relatively insensitive to an EGFR kinase inhibitor as asingle agent, and thus likely to show an enhanced response in thepresence of an mTOR inhibitor, and administering to said patientsimultaneously or sequentially a therapeutically effective amount of acombination of an EGFR kinase inhibitor and an mTOR inhibitor.

The present invention also provides a method for treating NSCL,pancreatic, colon or breast cancer tumors or tumor metastases in apatient, comprising the steps of diagnosing a patient's likelyresponsiveness to an EGFR kinase inhibitor by assessing whether thetumor cells have undergone an epithelial-mesenchymal transition,identifying the patient as one whose tumor or tumor metastases cellshave not undergone an epithelial-mesenchymal transition and are thuspredicted to be relatively sensitive to an EGFR kinase inhibitor as asingle agent, and may thus show an enhanced response in the presence ofan mTOR inhibitor, and administering to said patient simultaneously orsequentially a therapeutically effective amount of a combination of anEGFR kinase inhibitor and an mTOR inhibitor.

In a further embodiment of the above methods, the patient to be treatedis refractory to treatment with an EGFR kinase inhibitor as a singleagent. Thus, for example, in one embodiment, the present inventionprovides a method for treating NSCL, pancreatic, colon or breast cancertumors or tumor metastases in a patient refractory to treatment with anEGFR kinase inhibitor as a single agent, comprising administering tosaid patient simultaneously or sequentially a therapeutically effectiveamount of a combination of an EGFR kinase inhibitor and an mTORinhibitor. In an alternative embodiment, the present invention providesa method for treating NSCL, pancreatic, colon or breast cancer tumors ortumor metastases in a patient refractory to treatment with an EGFRkinase inhibitor as a single agent, comprising the steps of diagnosing apatient's likely responsiveness to an EGFR kinase inhibitor, andadministering to said patient simultaneously or sequentially atherapeutically effective amount of a combination of an EGFR kinaseinhibitor and an mTOR inhibitor. It will be appreciated by one of skillin the medical arts that there are many reasons for why a patient may berefractory to treatment with an EGFR kinase inhibitor as a single agent,one of which is that the tumor cells of the patient are relativelyinsensitive to inhibition by the tested EGFR kinase inhibitor. It isalso possible that a patient may be refractory to treatment with onetype of EGFR kinase inhibitor, but be sensitive to treatment withanother type of EGFR kinase inhibitor.

This invention also provides a method for treating abnormal cell growthof lung, pancreatic, colon or breast cancer cells in a patient,comprising administering to said patient simultaneously or sequentiallya therapeutically effective amount of a combination of an EGFR kinaseinhibitor and an mTOR inhibitor.

It will be appreciated by one of skill in the medical arts that theexact manner of administering to said patient of a therapeuticallyeffective amount of a combination of an EGFR kinase inhibitor and mTORinhibitor following a diagnosis of a patient's likely responsiveness toan EGFR kinase inhibitor will be at the discretion of the attendingphysician. The mode of administration, including dosage, combinationwith other anti-cancer agents, timing and frequency of administration,and the like, may be affected by the diagnosis of a patient's likelyresponsiveness to an EGFR kinase inhibitor, as well as the patient'scondition and history. Thus, even patients diagnosed with tumorspredicted to be relatively sensitive to an EGFR kinase inhibitor as asingle agent may still benefit from treatment with a combination of anEGFR kinase inhibitor and mTOR inhibitor, particularly in combinationwith other anti-cancer agents, or other agents that may alter a tumor'ssensitivity to EGFR kinase inhibitors.

In one embodiment of the methods of this invention, an mTOR inhibitor isadministered at the same time as the EGFR kinase inhibitor. In anotherembodiment of the methods of this invention, an mTOR inhibitor isadministered prior to the EGFR kinase inhibitor. In another embodimentof the methods of this invention, an mTOR inhibitor is administeredafter the EGFR kinase inhibitor. In another embodiment of the methods ofthis invention, an mTOR inhibitor is pre-administered prior toadministration of a combination of an EGFR kinase inhibitor and mTORinhibitor.

The present invention further provides a method for treating NSCL,pancreatic, colon or breast cancer tumors or tumor metastases in apatient, comprising administering to the patient simultaneously orsequentially a therapeutically effective amount of a combination of anEGFR kinase inhibitor and an agent that sensitizes tumor cells to theeffects of EGFR kinase inhibitors, and in addition, one or more othercytotoxic, chemotherapeutic or anti-cancer agents, or compounds thatenhance the effects of such agents.

In the context of this invention, other cytotoxic, chemotherapeutic oranti-cancer agents, or compounds that enhance the effects of suchagents, include, for example: alkylating agents or agents with analkylating action, such as cyclophosphamide (CTX; e.g. CYTOXAN®),chlorambucil (CHL; e.g. LEUKERAN®), cisplatin (C is P; e.g. PLATINOL®)busulfan (e.g. MYLERAN®), melphalan, carmustine (BCNU), streptozotocin,triethylenemelamine (TEM), mitomycin C, and the like; anti-metabolites,such as methotrexate (MTX), etoposide (VP16; e.g. VEPESID®),6-mercaptopurine (6MP), 6-thiocguanine (6TG), cytarabine (Ara-C),5-fluorouracil (5-FU), capecitabine (e.g. XELODA®), dacarbazine (DTIC),and the like; antibiotics, such as actinomycin D, doxorubicin (DXR; e.g.ADRIAMYCIN®), daunorubicin (daunomycin), bleomycin, mithramycin and thelike; alkaloids, such as vinca alkaloids such as vincristine (VCR),vinblastine, and the like; and other antitumor agents, such aspaclitaxel (e.g. TAXOL®) and pactitaxel derivatives, the cytostaticagents, glucocorticoids such as dexamethasone (DEX; e.g. DECADRON®) andcorticosteroids such as prednisone, nucleoside enzyme inhibitors such ashydroxyurea, amino acid depleting enzymes such as asparaginase,leucovorin and other folic acid derivatives, and similar, diverseantitumor agents. The following agents may also be used as additionalagents: amifostine (e.g. ETHYOL®), dactinomycin, mechlorethamine(nitrogen mustard), streptozocin, cyclophosphamide, lomustine (CCNU),doxorubicin lipo (e.g. DOXIL®), gemcitabine (e.g. GEMZAR®), daunorubicinlipo (e.g. DAUNOXOME®), procarbazine, mitomycin, docetaxel (e.g.TAXOTERE®), aldesleukin, carboplatin, oxaliplatin, cladribine,camptothecin, CPT 11 (irinotecan), 10-hydroxy 7-ethyl-camptothecin(SN38), floxuridine, fludarabine, ifosfamide, idarubicin, mesna,interferon beta, interferon alpha, mitoxantrone, topotecan, leuprolide,megestrol, melphalan, mercaptopurine, plicamycin, mitotane,pegaspargase, pentostatin, pipobroman, plicamycin, tamoxifen,teniposide, testolactone, thioguanine, thiotepa, uracil mustard,vinorelbine, chlorambucil.

The present invention further provides a method for treating NSCL,pancreatic, colon or breast cancer tumors or tumor metastases in apatient, comprising administering to said patient simultaneously orsequentially a therapeutically effective amount of a combination of anEGFR kinase inhibitor and an agent that sensitizes tumor cells to theeffects of EGFR kinase inhibitors, and in addition, one or moreanti-hormonal agents. As used herein, the term “anti-hormonal agent”includes natural or synthetic organic or peptidic compounds that act toregulate or inhibit hormone action on tumors.

Antihormonal agents include, for example: steroid receptor antagonists,anti-estrogens such as tamoxifen, raloxifene, aromatase inhibiting4(5)-imidazoles, other aromatase inhibitors, 42-hydroxytamoxifen,trioxifene, keoxifene, LY 117018, onapristone, and toremifene (e.g.FARESTON®); anti-androgens such as flutamide, nilutamide, bicalutamide,leuprolide, and goserelin; and pharmaceutically acceptable salts, acidsor derivatives of any of the above; agonists and/or antagonists ofglycoprotein hormones such as follicle stimulating hormone (FSH),thyroid stimulating hormone (TSH), and luteinizing hormone (LH) and LHRH(leuteinizing hormone-releasing hormone); the LHRH agonist goserelinacetate, commercially available as ZOLADEX® (AstraZeneca); the LHRHantagonist D-alaninamideN-acetyl-3-(2-naphthalenyl)-D-alanyl-4-chloro-D-phenylalanyl-3-(3-pyridinyl)-D-alanyl-L-seryl-N-6-(3-pyridinylcarbonyl)-L-lysyl-N-6-(3-pyridinylcarbonyl)-D-lysyl-L-leucyl-N-6-(1-methylethyl)-L-lysyl-L-proline(e.g ANTIDE®, Ares-Serono); the LHRH antagonist ganirelix acetate; thesteroidal anti-androgens cyproterone acetate (CPA) and megestrolacetate, commercially available as MEGACE® (Bristol-Myers Oncology); thenonsteroidal anti-androgenflutamide(2-methyl-N-[4,20-nitro-3-(trifluoromethyl)phenylpropanamide),commercially available as EULEXIN® (Schering Corp.); the non-steroidalanti-androgen nilutamide,(5,5-dimethyl-3-[4-nitro-3-(trifluoromethyl-4′-nitrophenyl)-4,4-dimethyl-imidazolidine-dione);and antagonists for other non-permissive receptors, such as antagonistsfor RAR, RXR, TR, VDR, and the like.

The use of the cytotoxic and other anticancer agents described above inchemotherapeutic regimens is generally well characterized in the cancertherapy arts, and their use herein falls under the same considerationsfor monitoring tolerance and effectiveness and for controllingadministration routes and dosages, with some adjustments. For example,the actual dosages of the cytotoxic agents may vary depending upon thepatient's cultured cell response determined by using histoculturemethods. Generally, the dosage will be reduced compared to the amountused in the absence of additional other agents.

Typical dosages of an effective cytotoxic agent can be in the rangesrecommended by the manufacturer, and where indicated by in vitroresponses or responses in animal models, can be reduced by up to aboutone order of magnitude concentration or amount. Thus, the actual dosagewill depend upon the judgment of the physician, the condition of thepatient, and the effectiveness of the therapeutic method based on the invitro responsiveness of the primary cultured malignant cells orhistocultured tissue sample, or the responses observed in theappropriate animal models.

The present invention further provides a method for treating NSCL,pancreatic, colon or breast cancer tumors or tumor metastases in apatient, comprising administering to said patient simultaneously orsequentially a therapeutically effective amount of a combination of anEGFR kinase inhibitor and an agent that sensitizes tumor cells to theeffects of EGFR kinase inhibitors, and in addition one or moreangiogenesis inhibitors.

Anti-angiogenic agents include, for example: VEGFR inhibitors, such asSU-5416 and SU-6668 (Sugen Inc. of South San Francisco, Calif., USA), oras described in, for example International Application Nos. WO 99/24440,WO 99/62890, WO 95/21613, WO 99/61422, WO 98/50356, WO 99/10349, WO97/32856, WO 97/22596, WO 98/54093, WO 98/02438, WO 99/16755, and WO98/02437, and U.S. Pat. Nos. 5,883,113, 5,886,020, 5,792,783, 5,834,504and 6,235,764; VEGF inhibitors such as IM862 (Cytran Inc. of Kirkland,Wash., USA); angiozyme, a synthetic ribozyme from Ribozyme (Boulder,Colo.) and Chiron (Emeryville, Calif.); and antibodies to VEGF, such asbevacizumab (e.g. AVASTIN™, Genentech, South San Francisco, Calif.), arecombinant humanized antibody to VEGF; integrin receptor antagonistsand integrin antagonists, such as to α_(v)β₃, α_(v)β₅ and α_(v)β₆integrins, and subtypes thereof, e.g. cilengitide (EMD 121974), or theanti-integrin antibodies, such as for example α_(v)β₃ specific humanizedantibodies (e.g. VITAXIN®); factors such as IFN-alpha (U.S. Pat. Nos.41,530,901, 4,503,035, and 5,231,176); angiostatin and plasminogenfragments (e.g. kringle 1-4, kringle 5, kringle 1-3 (O'Reilly, M. S. etal. (1994) Cell 79:315-328; Cao et al. (1996) J. Biol. Chem. 271:29461-29467; Cao et al. (1997) J. Biol. Chem. 272:22924-22928);endostatin (O'Reilly, M. S. et al. (1997) Cell 88:277; and InternationalPatent Publication No. WO 97/15666); thrombospondin (TSP-1; Frazier,(1991) Curr. Opin. Cell Biol. 3:792); platelet factor 4 (PF4);plasminogen activator/urokinase inhibitors; urokinase receptorantagonists; heparinases; fumagillin analogs such as TNP-4701; suraminand suramin analogs; angiostatic steroids; bFGF antagonists; flk-1 andfit-1 antagonists; anti-angiogenesis agents such as MMP-2(matrix-metalloproteinase 2) inhibitors and MMP-9(matrix-metalloproteinase 9) inhibitors. Examples of useful matrixmetalloproteinase inhibitors are described in International PatentPublication Nos. WO 96/33172, WO 96/27583, WO 98/07697, WO 98/03516, WO98/34918, WO 98/34915, WO 98/33768, WO 98/30566, WO 90/05719, WO99/52910, WO 99/52889, WO 99/29667, and WO 99/07675, European PatentPublication Nos. 818,442, 780,386, 1,004,578, 606,046, and 931,788;Great Britain Patent Publication No. 9912961, and U.S. Pat. Nos.5,863,949 and 5,861,510. Preferred MMP-2 and MMP-9 inhibitors are thosethat have little or no activity inhibiting MMP-1. More preferred, arethose that selectively inhibit MMP-2 and/or MMP-9 relative to the othermatrix-metalloproteinases (i.e. MMP-1, MMP-3, MMP-4, MMP-5, MMP-6,MMP-7, MMP-8, MMP-10, MMP-11, MMP-12, and MMP-13).

The present invention further provides a method for treating NSCL,pancreatic, colon or breast cancer tumors or tumor metastases in apatient, comprising administering to the patient simultaneously orsequentially a therapeutically effective amount of a combination of anEGFR kinase inhibitor and an agent that sensitizes tumor cells to theeffects of EGFR kinase inhibitors, and in addition one or more tumorcell pro-apoptotic or apoptosis-stimulating agents.

The present invention further provides a method for treating NSCL,pancreatic, colon or breast cancer tumors or tumor metastases in apatient, comprising administering to said patient simultaneously orsequentially a therapeutically effective amount of a combination of anEGFR kinase inhibitor and an agent that sensitizes tumor cells to theeffects of EGFR kinase inhibitors, and in addition one or more signaltransduction inhibitors.

Signal transduction inhibitors include, for example: erbB2 receptorinhibitors, such as organic molecules, or antibodies that bind to theerbB2 receptor, for example, trastuzumab (e.g. HERCEPTIN®); inhibitorsof other protein tyrosine-kinases, e.g. imitinib (e.g. GLEEVEC®); rasinhibitors; raf inhibitors; MEK inhibitors; mTOR inhibitors; cyclindependent kinase inhibitors; protein kinase C inhibitors; and PDK-1inhibitors (see Dancey, J. and Sausville, E. A. (2003) Nature Rev. DrugDiscovery 2:92-313, for a description of several examples of suchinhibitors, and their use in clinical trials for the treatment ofcancer).

ErbB2 receptor inhibitors include, for example: ErbB2 receptorinhibitors, such as GW-282974 (Glaxo Wellcome plc), monoclonalantibodies such as AR-209 (Aronex Pharmaceuticals Inc. of The Woodlands,Tex., USA) and 2B-1 (Chiron), and erbB2 inhibitors such as thosedescribed in International Publication Nos. WO 98/02434, WO 99/35146, WO99/35132, WO 98/02437, WO 97/13760, and WO 95/19970, and U.S. Pat. Nos.5,587,458, 5,877,305, 6,465,449 and 6,541,481.

The present invention further thus provides a method for treating NSCL,pancreatic, colon or breast cancer tumors or tumor metastases in apatient, comprising administering to said patient simultaneously orsequentially a therapeutically effective amount of a combination of anEGFR kinase inhibitor and an agent that sensitizes tumor cells to theeffects of EGFR kinase inhibitors, and in addition an anti-HER2 antibodyor an immunotherapeutically active fragment thereof.

The present invention further provides a method for treating NSCL,pancreatic, colon or breast cancer tumors or tumor metastases in apatient, comprising administering to said patient simultaneously orsequentially a therapeutically effective amount of a combination of anEGFR kinase inhibitor and an agent that sensitizes tumor cells to theeffects of EGFR kinase inhibitors, and in addition one or moreadditional anti-proliferative agents.

Additional antiproliferative agents include, for example: Inhibitors ofthe enzyme farnesyl protein transferase and inhibitors of the receptortyrosine kinase PDGFR, including the compounds disclosed and claimed inU.S. Pat. Nos. 6,080,769, 6,194,438, 6,258,824, 6,586,447, 6,071,935,6,495,564, 6,150,377, 6,596,735 and 6,479,513, and International PatentPublication WO 01/40217.

The present invention further provides a method for treating NSCL,pancreatic, colon or breast cancer tumors or tumor metastases in apatient, comprising administering to the patient simultaneously orsequentially a therapeutically effective amount of a combination of anEGFR kinase inhibitor and an agent that sensitizes tumor cells to theeffects of EGFR kinase inhibitors, and in addition a COX II(cyclooxygenase II) inhibitor. Examples of useful COX-II inhibitorsinclude alecoxib (e.g. CELEBREX™), valdecoxib, and rofecoxib.

The present invention further provides a method for treating NSCL,pancreatic, colon or breast cancer tumors or tumor metastases in apatient, comprising administering to the patient simultaneously orsequentially a therapeutically effective amount of a combination of anEGFR kinase inhibitor and an agent that sensitizes tumor cells to theeffects of EGFR kinase inhibitors, and in addition treatment withradiation or a radiopharmaceutical.

The source of radiation can be either external or internal to thepatient being treated. When the source is external to the patient, thetherapy is known as external beam radiation therapy (EBRT). When thesource of radiation is internal to the patient, the treatment is calledbrachytherapy (BT). Radioactive atoms for use in the context of thisinvention can be selected from the group including, but not limited to,radium, cesium-137, iridium-192, americium-241, gold-198, cobalt-57,copper-67, technetium-99, iodine-123, iodine-131, and indium-111. Wherethe EGFR kinase inhibitor according to this invention is an antibody, itis also possible to label the antibody with such radioactive isotopes.

Radiation therapy is a standard treatment for controlling unresectableor inoperable tumors and/or tumor metastases. Improved results have beenseen when radiation therapy has been combined with chemotherapy.Radiation therapy is based on the principle that high-dose radiationdelivered to a target area will result in the death of reproductivecells in both tumor and normal tissues. The radiation dosage regimen isgenerally defined in terms of radiation absorbed dose (Gy), time andfractionation, and must be carefully defined by the oncologist. Theamount of radiation a patient receives will depend on variousconsiderations, but the two most important are the location of the tumorin relation to other critical structures or organs of the body, and theextent to which the tumor has spread. A typical course of treatment fora patient undergoing radiation therapy will be a treatment schedule overa 1 to 6 week period, with a total dose of between 10 and 80 Gyadministered to the patient in a single daily fraction of about 1.8 to2.0 Gy, 5 days a week. In a preferred embodiment of this invention thereis synergy when tumors in human patients are treated with thecombination treatment of the invention and radiation. In other words,the inhibition of tumor growth by means of the agents comprising thecombination of the invention is enhanced when combined with radiation,optionally with additional chemotherapeutic or anticancer agents.Parameters of adjuvant radiation therapies are, for example, containedin International Patent Publication WO 99/60023.

The present invention further provides a method for treating NSCL,pancreatic, colon or breast cancer tumors or tumor metastases in apatient, comprising administering to the patient simultaneously orsequentially a therapeutically effective amount of a combination of anEGFR kinase inhibitor and an agent that sensitizes tumor cells to theeffects of EGFR kinase inhibitors, and in addition treatment with one ormore agents capable of enhancing antitumor immune responses.

Agents capable of enhancing antitumor immune responses include, forexample: CTLA4 (cytotoxic lymphocyte antigen 4) antibodies (e.g.MDX-CTLA4), and other agents capable of blocking CTLA4. Specific CTLA4antibodies that can be used in the present invention include thosedescribed in U.S. Pat. No. 6,682,736.

The present invention further provides a method for reducing the sideeffects caused by the treatment of NSCL, pancreatic, colon or breastcancer tumors or tumor metastases in a patient with an EGFR kinaseinhibitor or an mTOR inhibitor, comprising administering to the patientsimultaneously or sequentially a therapeutically effective amount of acombination of an EGFR kinase inhibitor and an agent that sensitizestumor cells to the effects of EGFR kinase inhibitors (i.e. an mTORinhibitor), in amounts that are effective to produce an additive, or asuperadditive or synergistic antitumor effect, and that are effective atinhibiting the growth of the tumor.

The present invention further provides a method for the treatment ofNSCL, pancreatic, colon or breast cancer, comprising administering to asubject in need of such treatment (i) an effective first amount of anEGFR kinase inhibitor, or a pharmaceutically acceptable salt thereof;and (ii) an effective second amount of an agent that sensitizes tumorcells to the effects of EGFR kinase inhibitors.

The present invention also provides a method for the treatment of NSCL,pancreatic, colon or breast cancer, comprising administering to asubject in need of such treatment (i) a sub-therapeutic first amount ofan EGFR kinase inhibitor, or a pharmaceutically acceptable salt thereof;and (ii) a sub-therapeutic second amount of an agent that sensitizestumor cells to the effects of EGFR kinase inhibitors.

The present invention also provides a method for the treatment of NSCL,pancreatic, colon or breast cancer, comprising administering to asubject in need of such treatment (i) an effective first amount of anEGFR kinase inhibitor, or a pharmaceutically acceptable salt thereof;and (ii) a sub-therapeutic second amount of an agent that sensitizestumor cells to the effects of EGFR kinase inhibitors.

The present invention also provides a method for the treatment of NSCL,pancreatic, colon or breast cancer, comprising administering to asubject in need of such treatment (i) a sub-therapeutic first amount ofan EGFR kinase inhibitor, or a pharmaceutically acceptable salt thereof;and (ii) an effective second amount of an agent that sensitizes tumorcells to the effects of EGFR kinase inhibitors.

In the preceding methods the order of administration of the first andsecond amounts can be simultaneous or sequential, i.e. the agent thatsensitizes tumor cells to the effects of EGFR kinase inhibitors can beadministered before the EGFR kinase inhibitor, after the EGFR inhibitor,or at the same time as the EGFR kinase inhibitor. In an alternativeembodiment of each of these methods, the NSCL, pancreatic, colon orbreast cancer has low sensitivity or is relatively insensitive orrefractory to inhibition by EGFR kinase inhibitors such as erlotinib assingle agents.

In the context of this invention, an “effective amount” of an agent ortherapy is as defined above. A “sub-therapeutic amount” of an agent ortherapy is an amount less than the effective amount for that agent ortherapy, but when combined with an effective or sub-therapeutic amountof another agent or therapy can produce a result desired by thephysician, due to, for example, synergy in the resulting efficaciouseffects, or reduced side effects.

Additionally, the present invention provides a pharmaceuticalcomposition comprising a combination of an EGFR kinase inhibitor and anagent that sensitizes tumor cells to the effects of EGFR kinaseinhibitors in a pharmaceutically acceptable carrier.

As used herein, the term “patient” preferably refers to a human in needof treatment with an EGFR kinase inhibitor for any purpose, and morepreferably a human in need of such a treatment to treat cancer, or aprecancerous condition or lesion. However, the term “patient” can alsorefer to non-human animals, preferably mammals such as dogs, cats,horses, cows, pigs, sheep and non-human primates, among others, that arein need of treatment with an EGFR kinase inhibitor.

In a preferred embodiment, the patient is a human in need of treatmentfor cancer, or a precancerous condition or lesion, wherein the cancer ispreferably NSCL, breast, colon or pancreatic cancer. In addition, othercancers that may be treated by the methods described herein includeexamples of the following cancers that are treatable by administrationof an EGFR kinase inhibitor: lung cancer, bronchioloalveolar cell lungcancer, bone cancer, skin cancer, cancer of the head or neck, cutaneousor intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer,cancer of the anal region, stomach cancer, gastric cancer, uterinecancer, carcinoma of the fallopian tubes, carcinoma of the endometrium,carcinoma of the vagina, carcinoma of the vulva, Hodgkin's Disease,cancer of the esophagus, cancer of the small intestine, cancer of theendocrine system, cancer of the thyroid gland, cancer of the parathyroidgland, cancer of the adrenal gland, sarcoma of soft tissue, cancer ofthe urethra, cancer of the penis, prostate cancer, cancer of thebladder, cancer of the ureter, carcinoma of the renal pelvis,mesothelioma, hepatocellular cancer, biliary cancer, chronic or acuteleukemia, lymphocytic lymphomas, neoplasms of the central nervous system(CNS), spinal axis tumors, brain stem glioma, glioblastoma multiforme,astrocytomas, schwannomas, ependymomas, medulloblastomas, meningiomas,squamous cell carcinomas, pituitary adenomas, including refractoryversions of any of the above cancers, or a combination of one or more ofthe above cancers. The precancerous condition or lesion includes, forexample, the group consisting of oral leukoplakia, actinic keratosis(solar keratosis), precancerous polyps of the colon or rectum, gastricepithelial dyspiasia, adenomatous dysplasia, hereditary nonpolyposiscolon cancer syndrome (HNPCC), Barrett's esophagus, bladder dysplasia,and precancerous cervical conditions. In addition, other cancers thatmay be treated by the methods described herein include examples of thefollowing cancers that are treatable by administration of the EGFRkinase inhibitor erlotinib: cancer of the kidney or renal cellcarcinoma.

The term “refractory” as used herein is used to define a cancer forwhich treatment (e.g. chemotherapy drugs, biological agents, and/orradiation therapy) has proven to be ineffective. A refractory cancertumor may shrink, but not to the point where the treatment is determinedto be effective. Typically however, the tumor stays the same size as itwas before treatment (stable disease), or it grows (progressivedisease).

For purposes of the present invention, “co-administration of” and“co-administering” an EGFR kinase inhibitor and an agent that sensitizestumor cells to the effects of EGFR kinase inhibitors (both componentsreferred to hereinafter as the “two active agents”) refer to anyadministration of the two active agents, either separately or together,where the two active agents are administered as part of an appropriatedose regimen designed to obtain the benefit of the combination therapy.Thus, the two active agents can be administered either as part of thesame pharmaceutical composition or in separate pharmaceuticalcompositions. The agent that sensitizes tumor cells to the effects ofEGFR kinase inhibitors can be administered prior to, at the same timeas, or subsequent to administration of the EGFR kinase inhibitor, or insome combination thereof. Where the EGFR kinase inhibitor isadministered to the patient at repeated intervals, e.g., during astandard course of treatment, the agent that sensitizes tumor cells tothe effects of EGFR kinase inhibitors can be administered prior to, atthe same time as, or subsequent to, each administration of the EGFRkinase inhibitor, or some combination thereof, or at different intervalsin relation to the EGFR kinase inhibitor treatment, or in a single doseprior to, at any time during, or subsequent to the course of treatmentwith the EGFR kinase inhibitor.

The EGFR kinase inhibitor will typically be administered to the patientin a dose regimen that provides for the most effective treatment of thecancer (from both efficacy and safety perspectives) for which thepatient is being treated, as known in the art, and as disclosed, e.g. inInternational Patent Publication No. WO 01/34574. In conducting thetreatment method of the present invention, the EGFR kinase inhibitor canbe administered in any effective manner known in the art, such as byoral, topical, intravenous, intra-peritoneal, intramuscular,intra-articular, subcutaneous, intranasal, intra-ocular, vaginal,rectal, or intradermal routes, depending upon the type of cancer beingtreated, the type of EGFR kinase inhibitor being used (for example,small molecule, antibody, RNAi, ribozyme or antisense construct), andthe medical judgement of the prescribing physician as based, e.g., onthe results of published clinical studies.

The amount of EGFR kinase inhibitor administered and the timing of EGFRkinase inhibitor administration will depend on the type (species,gender, age, weight, etc.) and condition of the patient being treated,the severity of the disease or condition being treated, and on the routeof administration. For example, small molecule EGFR kinase inhibitorscan be administered to a patient in doses ranging from 0.001 to 100mg/kg of body weight per day or per week in single or divided doses, orby continuous infusion (see for example, International PatentPublication No. WO 01/34574). In particular, erlotinib HCl can beadministered to a patient in doses ranging from 5-200 mg per day, or100-1600 mg per week, in single or divided doses, or by continuousinfusion. A preferred dose is 150 mg/day. Antibody-based EGFR kinaseinhibitors, or antisense, RNAi or ribozyme constructs, can beadministered to a patient in doses ranging from 0.1 to 100 mg/kg of bodyweight per day or per week in single or divided doses, or by continuousinfusion. In some instances, dosage levels below the lower limit of theaforesaid range may be more than adequate, while in other cases stilllarger doses may be employed without causing any harmful side effect,provided that such larger doses are first divided into several smalldoses for administration throughout the day.

The EGFR kinase inhibitors and the agent that sensitizes tumor cells tothe effects of EGFR kinase inhibitors can be administered eitherseparately or together by the same or different routes, and in a widevariety of different dosage forms. For example, the EGFR kinaseinhibitor is preferably administered orally or parenterally. The agentthat sensitizes tumor cells to the effects of EGFR kinase inhibitors ispreferably administered orally or parenterally. Where the EGFR kinaseinhibitor is erlotinib HCl (TARCEVA®), oral administration ispreferable. Both the EGFR kinase inhibitors and the agent thatsensitizes tumor cells to the effects of EGFR kinase inhibitors can beadministered in single or multiple doses. In one embodiment, the agentthat sensitizes tumor cells to the effects of EGFR kinase inhibitors isadministered first as a pretreatment, followed by administration of thecombination of both agents (EGFR kinase inhibitor and the agent thatsensitizes tumor cells to the effects of EGFR kinase inhibitors), eitherseparately or combined together in one formulation.

The EGFR kinase inhibitor can be administered with variouspharmaceutically acceptable inert carriers in the form of tablets,capsules, lozenges, troches, hard candies, powders, sprays, creams,salves, suppositories, jellies, gels, pastes, lotions, ointments,elixirs, syrups, and the like. Administration of such dosage forms canbe carried out in single or multiple doses. Carriers include soliddiluents or fillers, sterile aqueous media and various non-toxic organicsolvents, etc. Oral pharmaceutical compositions can be suitablysweetened and/or flavored.

The EGFR kinase inhibitor and the agent that sensitizes tumor cells tothe effects of EGFR kinase inhibitors can be combined together withvarious pharmaceutically acceptable inert carriers in the form ofsprays, creams, salves, suppositories, jellies, gels, pastes, lotions,ointments, and the like. Administration of such dosage forms can becarried out in single or multiple doses. Carriers include solid diluentsor fillers, sterile aqueous media, and various non-toxic organicsolvents, etc.

All formulations comprising proteinaceous EGFR kinase inhibitors shouldbe selected so as to avoid denaturation and/or degradation and loss ofbiological activity of the inhibitor.

Methods of preparing pharmaceutical compositions comprising an EGFRkinase inhibitor are known in the art, and are described, e.g. inInternational Patent Publication No. WO 01/34574. Methods of preparingpharmaceutical compositions comprising mTOR inhibitors are also wellknown in the art (e.g. see International Patent Publication WO2004/004644, or patents on rapamycin macrolides referred to therein). Inview of the teaching of the present invention, methods of preparingpharmaceutical compositions comprising both an EGFR kinase inhibitor andthe agent that sensitizes tumor cells to the effects of EGFR kinaseinhibitors will be apparent from the above-cited publications and fromother known references, such as Remington's Pharmaceutical Sciences,Mack Publishing Company, Easton, Pa., 18^(th) edition (1990).

For oral administration of EGFR kinase inhibitors, tablets containingone or both of the active agents are combined with any of variousexcipients such as, for example, micro-crystalline cellulose, sodiumcitrate, calcium carbonate, dicalcium phosphate and glycine, along withvarious disintegrants such as starch (and preferably corn, potato ortapioca starch), alginic acid and certain complex silicates, togetherwith granulation binders like polyvinyl pyrrolidone, sucrose, gelatinand acacia. Additionally, lubricating agents such as magnesium stearate,sodium lauryl sulfate and talc are often very useful for tabletingpurposes. Solid compositions of a similar type may also be employed asfillers in gelatin capsules; preferred materials in this connection alsoinclude lactose or milk sugar as well as high molecular weightpolyethylene glycols. When aqueous suspensions and/or elixirs aredesired for oral administration, the EGFR kinase inhibitor may becombined with various sweetening or flavoring agents, coloring matter ordyes, and, if so desired, emulsifying and/or suspending agents as well,together with such diluents as water, ethanol, propylene glycol,glycerin and various like combinations thereof.

For parenteral administration of either or both of the active agents,solutions in either sesame or peanut oil or in aqueous propylene glycolmay be employed, as well as sterile aqueous solutions comprising theactive agent or a corresponding water-soluble salt thereof. Such sterileaqueous solutions are preferably suitably buffered, and are alsopreferably rendered isotonic, e.g., with sufficient saline or glucose.These particular aqueous solutions are especially suitable forintravenous, intramuscular, subcutaneous and intraperitoneal injectionpurposes. The oily solutions are suitable for intra-articular,intramuscular and subcutaneous injection purposes. The preparation ofall these solutions under sterile conditions is readily accomplished bystandard pharmaceutical techniques well known to those skilled in theart. Any parenteral formulation selected for administration ofproteinaceous EGFR kinase inhibitors should be selected so as to avoiddenaturation and loss of biological activity of the inhibitor.

Additionally, it is possible to topically administer either or both ofthe active agents, by way of, for example, creams, lotions, jellies,gels, pastes, ointments, salves and the like, in accordance withstandard pharmaceutical practice. For example, a topical formulationcomprising either an EGFR kinase inhibitor or the agent that sensitizestumor cells to the effects of EGFR kinase inhibitors in about 0.1% (w/v)to about 5% (w/v) concentration can be prepared.

For veterinary purposes, the active agents can be administeredseparately or together to animals using any of the forms and by any ofthe routes described above. In a preferred embodiment, the EGFR kinaseinhibitor is administered in the form of a capsule, bolus, tablet,liquid drench, by injection or as an implant. As an alternative, theEGFR kinase inhibitor can be administered with the animal feedstuff, andfor this purpose a concentrated feed additive or premix may be preparedfor a normal animal feed. The agent that sensitizes tumor cells to theeffects of EGFR kinase inhibitors is preferably administered in the formof liquid drench, by injection or as an implant. Such formulations areprepared in a conventional manner in accordance with standard veterinarypractice.

The present invention further provides a kit comprising a singlecontainer comprising both an EGFR kinase inhibitor and the agent thatsensitizes tumor cells to the effects of EGFR kinase inhibitors. Thepresent invention further provides a kit comprising a first containercomprising an EGFR kinase inhibitor and a second container comprisingthe agent that sensitizes tumor cells to the effects of EGFR kinaseinhibitors. In a preferred embodiment, the kit containers may furtherinclude a pharmaceutically acceptable carrier. The kit may furtherinclude a sterile diluent, which is preferably stored in a separateadditional container. The kit may further include a package insertcomprising printed instructions directing the use of the combinedtreatment as a method for treating cancer. The kit may also compriseadditional containers comprising additional anti-cancer agents, agentsthat enhances the effect of such agents, or other compounds that improvethe efficacy or tolerability of the treatment.

As used herein, the term “EGFR kinase inhibitor” refers to any EGFRkinase inhibitor that is currently known in the art or that will beidentified in the future, and includes any chemical entity that, uponadministration to a patient, results in inhibition of a biologicalactivity associated with activation of the EGF receptor in the patient,including any of the downstream biological effects otherwise resultingfrom the binding to EGFR of its natural ligand. Such EGFR kinaseinhibitors include any agent that can block EGFR activation or any ofthe downstream biological effects of EGFR activation that are relevantto treating cancer in a patient. Such an inhibitor can act by bindingdirectly to the intracellular domain of the receptor and inhibiting itskinase activity. Alternatively, such an inhibitor can act by occupyingthe ligand binding site or a portion thereof of the EGF receptor,thereby making the receptor inaccessible to its natural ligand so thatits normal biological activity is prevented or reduced. Alternatively,such an inhibitor can act by modulating the dimerization of EGFRpolypeptides, or interaction of EGFR polypeptide with other proteins, orenhance ubiquitination and endocytotic degradation of EGFR. EGFR kinaseinhibitors include but are not limited to low molecular weightinhibitors, antibodies or antibody fragments, peptide or RNA aptamers,antisense constructs, small inhibitory RNAs (i.e. RNA interference bydsRNA; RNAi), and ribozymes. In a preferred embodiment, the EGFR kinaseinhibitor is a small organic molecule or an antibody that bindsspecifically to the human EGFR.

EGFR kinase inhibitors include, for example quinazoline EGFR kinaseinhibitors, pyrido-pyrimidine EGFR kinase inhibitors,pyrimido-pyrimidine EGFR kinase inhibitors, pyrrolo-pyrimidine EGFRkinase inhibitors, pyrazolo-pyrimidine EGFR kinase inhibitors,phenylamino-pyrimidine EGFR kinase inhibitors, oxindole EGFR kinaseinhibitors, indolocarbazole EGFR kinase inhibitors, phthalazine EGFRkinase inhibitors, isoflavone EGFR kinase inhibitors, quinalone EGFRkinase inhibitors, and tyrphostin EGFR kinase inhibitors, such as thosedescribed in the following patent publications, and all pharmaceuticallyacceptable salts and solvates of said EGFR kinase inhibitors:International Patent Publication Nos. WO 96/33980, WO 96/30347, WO97/30034, WO 97/30044, WO 97/38994, WO 97/49688, WO 98/02434, WO97/38983, WO 95/19774, WO 95/19970, WO 97/13771, WO 98/02437, WO98/02438, WO 97/32881, WO 98/33798, WO 97/32880, WO 97/3288, WO97/02266, WO 97/27199, WO 98/07726, WO 97/34895, WO 96/31510, WO98/14449, WO 98/14450, WO 98/14451, WO 95/09847, WO 97/19065, WO98/17662, WO 99/35146, WO 99/35132, WO 99/07701, and WO 92/20642;European Patent Application Nos. EP 520722, EP 566226, EP 787772, EP837063, and EP 682027; U.S. Pat. Nos. 5,747,498, 5,789,427, 5,650,415,and 5,656,643; and German Patent Application No. DE 19629652. Additionalnon-limiting examples of low molecular weight EGFR kinase inhibitorsinclude any of the EGFR kinase inhibitors described in Traxler, P.,1998, Exp. Opin. Ther. Patents 8(12):1599-1625.

Specific preferred examples of low molecular weight EGFR kinaseinhibitors that can be used according to the present invention include[6,7-bis(2-methoxyethoxy)-4-quinazolin-4-yl]-(3-ethynylphenyl)amine(also known as OSI-774, erlotinib, or TARCEVA® (erlotinib HCl); OSIPharmaceuticals/Genentech/Roche) (U.S. Pat. No. 5,747,498; InternationalPatent Publication No. WO 01/34574, and Moyer, J. D. et al. (1997)Cancer Res. 57:4838-4848); CI-1033 (formerly known as PD183805; Pfizer)(Sherwood et al., 1999, Proc. Am. Assoc. Cancer Res. 40:723); PD-158780(Pfizer); AG-1478 (University of California); CGP-59326 (Novartis);PKI-166 (Novartis); EKB-569 (Wyeth); GW-2016 (also known as GW-572016 orlapatinib ditosylate; GSK); and gefitinib (also known as ZD1839 orIRESSA™; Astrazeneca) (Woodburn et al., 1997, Proc. Am. Assoc. CancerRes. 38:633). A particularly preferred low molecular weight EGFR kinaseinhibitor that can be used according to the present invention is[6,7-bis(2-methoxyethoxy)-4-quinazolin-4-yl]-(3-ethynylphenyl)amine(i.e. erlotinib), its hydrochloride salt (i.e. erlotinib HCl, TARCEVA®),or other salt forms (e.g. erlotinib mesylate).

EGFR kinase inhibitors also include, for example multi-kinase inhibitorsthat have activity on EGFR kinase, i.e. inhibitors that inhibit EGFRkinase and one or more additional kinases. Examples of such compoundsinclude the EGFR and HER2 inhibitor CI-1033 (formerly known as PD183805;Pfizer); the EGFR and HER2 inhibitor GW-2016 (also known as GW-572016 orlapatinib ditosylate; GSK); the EGFR and JAK 2/3 inhibitor AG490 (atyrphostin); the EGFR and HER2 inhibitor ARRY-334543 (Array BioPharma);BIBW-2992, an irreversible dual EGFR/HER2 kinase inhibitor (BoehringerIngelheim Corp.); the EGFR and HER2 inhibitor EKB-569 (Wyeth); theVEGF-R2 and EGFR inhibitor ZD6474 (also known as ZACTIMA™; AstraZenecaPharmaceuticals), and the EGFR and HER2 inhibitor BMS-599626(Bristol-Myers Squibb).

Antibody-based EGFR kinase inhibitors include any anti-EGFR antibody orantibody fragment that can partially or completely block EGFR activationby its natural ligand. Non-limiting examples of antibody-based EGFRkinase inhibitors include those described in Modjtahedi, H., et al.,1993, Br. J. Cancer 67:247-253; Teramoto, T., et al., 1996, Cancer77:639-645; Goldstein et al., 1995, Clin. Cancer Res. 1:1311-1318;Huang, S. M., et al., 1999, Cancer Res. 15:59(8):1935-40; and Yang, X.,et al., 1999, Cancer Res. 59:1236-1243. Thus, the EGFR kinase inhibitorcan be the monoclonal antibody Mab E7.6.3 (Yang, X. D. et al. (1999)Cancer Res. 59:1236-43), or Mab C225 (ATCC Accession No. HB-8508), or anantibody or antibody fragment having the binding specificity thereof.Suitable monoclonal antibody EGFR kinase inhibitors include, but are notlimited to, IMC-C225 (also known as cetuximab or ERBITUX™; ImcloneSystems), ABX-EGF (Abgenix), EMD 72000 (Merck KgaA, Darmstadt), RH3(York Medical Bioscience Inc.), and MDX-447 (Medarex/Merck KgaA).

Additional antibody-based EGFR kinase inhibitors can be raised accordingto known methods by administering the appropriate antigen or epitope toa host animal selected, e.g., from pigs, cows, horses, rabbits, goats,sheep, and mice, among others. Various adjuvants known in the art can beused to enhance antibody production.

Although antibodies useful in practicing the invention can bepolyclonal, monoclonal antibodies are preferred. Monoclonal antibodiesagainst EGFR can be prepared and isolated using any technique thatprovides for the production of antibody molecules by continuous celllines in culture. Techniques for production and isolation include butare not limited to the hybridoma technique originally described byKohler and Milstein (Nature, 1975, 256: 495-497); the human B-cellhybridoma technique (Kosbor et al., 1983, Immunology Today 4:72; Cote etal., 1983, Proc. Natl. Acad. Sci. USA 80: 2026-2030); and theEBV-hybridoma technique (Cole et al, 1985, Monoclonal Antibodies andCancer Therapy, Alan R. Liss, Inc., pp. 77-96).

Alternatively, techniques described for the production of single chainantibodies (see, e.g., U.S. Pat. No. 4,946,778) can be adapted toproduce anti-EGFR single chain antibodies. Antibody-based EGFR kinaseinhibitors useful in practicing the present invention also includeanti-EGFR antibody fragments including but not limited to F(ab′).sub.2fragments, which can be generated by pepsin digestion of an intactantibody molecule, and Fab fragments, which can be generated by reducingthe disulfide bridges of the F(ab′).sub.2 fragments. Alternatively, Faband/or scFv expression libraries can be constructed (see, e.g., Huse etal., 1989, Science 246: 1275-1281) to allow rapid identification offragments having the desired specificity to EGFR.

Techniques for the production and isolation of monoclonal antibodies andantibody fragments are well-known in the art, and are described inHarlow and Lane, 1988, Antibodies: A Laboratory Manual, Cold SpringHarbor Laboratory, and in J. W. Goding, 1986, Monoclonal Antibodies:Principles and Practice, Academic Press, London. Humanized anti-EGFRantibodies and antibody fragments can also be prepared according toknown techniques such as those described in Vaughn, T. J. et al., 1998,Nature Biotech. 16:535-539 and references cited therein, and suchantibodies or fragments thereof are also useful in practicing thepresent invention.

EGFR kinase inhibitors for use in the present invention canalternatively be peptide or RNA aptamers. Such aptamers can for exampleinteract with the extracellular or intracellular domains of EGFR toinhibit EGFR kinase activity in cells. An aptamer that interacts withthe extracellular domain is preferred as it would not be necessary forsuch an aptamer to cross the plasma membrane of the target cell. Anaptamer could also interact with the ligand for EGFR (e.g. EGF, TGF-),such that its ability to activate EGFR is inhibited. Methods forselecting an appropriate aptamer are well known in the art. Such methodshave been used to select both peptide and RNA aptamers that interactwith and inhibit EGFR family members (e.g. see Buerger, C. et al. et al.(2003) J. Biol. Chem. 278:37610-37621; Chen, C-H. B. et al. (2003) Proc.Natl. Acad. Sci. 100:9226-9231; Buerger, C. and Groner, B. (2003) J.Cancer Res. Clin. Oncol. 129(12):669-675. Epub 2003 Sep. 11.).

EGFR kinase inhibitors for use in the present invention canalternatively be based on antisense oligonucleotide constructs.Anti-sense oligonucleotides, including anti-sense RNA molecules andanti-sense DNA molecules, would act to directly block the translation ofEGFR mRNA by binding thereto and thus preventing protein translation orincreasing mRNA degradation, thus decreasing the level of EGFR kinaseprotein, and thus activity, in a cell. For example, antisenseoligonucleotides of at least about 15 bases and complementary to uniqueregions of the mRNA transcript sequence encoding EGFR can besynthesized, e.g., by conventional phosphodiester techniques andadministered by e.g., intravenous injection or infusion. Methods forusing antisense techniques for specifically inhibiting gene expressionof genes whose sequence is known are well known in the art (e.g. seeU.S. Pat. Nos. 6,566,135; 6,566,131; 6,365,354; 6,410,323; 6,107,091;6,046,321; and 5,981,732).

Small inhibitory RNAs (siRNAs) can also function as EGFR kinaseinhibitors for use in the present invention. EGFR gene expression can bereduced by contacting the tumor, subject or cell with a small doublestranded RNA (dsRNA), or a vector or construct causing the production ofa small double stranded RNA, such that expression of EGFR isspecifically inhibited (i.e. RNA interference or RNAi). Methods forselecting an appropriate dsRNA or dsRNA-encoding vector are well knownin the art for genes whose sequence is known (e.g. see Tuschi, T., etal. (1999) Genes Dev. 13(24):3191-3197; Elbashir, S. M. et al. (2001)Nature 411:494-498; Hannon, G. J. (2002) Nature 418:244-251; McManus, M.T. and Sharp, P. A. (2002) Nature Reviews Genetics 3:737-747;Bremmelkamp, T. R. et al. (2002) Science 296:550-553; U.S. Pat. Nos.6,573,099 and 6,506,559; and International Patent Publication Nos. WO01/36646, WO 99/32619, and WO 01/68836).

Ribozymes can also function as EGFR kinase inhibitors for use in thepresent invention. Ribozymes are enzymatic RNA molecules capable ofcatalyzing the specific cleavage of RNA. The mechanism of ribozymeaction involves sequence specific hybridization of the ribozyme moleculeto complementary target RNA, followed by endonucleolytic cleavage.Engineered hairpin or hammerhead motif ribozyme molecules thatspecifically and efficiently catalyze endonucleolytic cleavage of EGFRmRNA sequences are thereby useful within the scope of the presentinvention. Specific ribozyme cleavage sites within any potential RNAtarget are initially identified by scanning the target molecule forribozyme cleavage sites, which typically include the followingsequences, GUA, GUU, and GUC. Once identified, short RNA sequences ofbetween about 15 and 20 ribonucleotides corresponding to the region ofthe target gene containing the cleavage site can be evaluated forpredicted structural features, such as secondary structure, that canrender the oligonucleotide sequence unsuitable. The suitability ofcandidate targets can also be evaluated by testing their accessibilityto hybridization with complementary oligonucleotides, using, e.g.,ribonuclease protection assays.

Both antisense oligonucleotides and ribozymes useful as EGFR kinaseinhibitors can be prepared by known methods. These include techniquesfor chemical synthesis such as, e.g., by solid phase phosphoramaditechemical synthesis. Alternatively, anti-sense RNA molecules can begenerated by in vitro or in vivo transcription of DNA sequences encodingthe RNA molecule. Such DNA sequences can be incorporated into a widevariety of vectors that incorporate suitable RNA polymerase promoterssuch as the T7 or SP6 polymerase promoters. Various modifications to theoligonucleotides of the invention can be introduced as a means ofincreasing intracellular stability and half-life. Possible modificationsinclude but are not limited to the addition of flanking sequences ofribonucleotides or deoxyribonucleotides to the 5′ and/or 3′ ends of themolecule, or the use of phosphorothioate or 2′-O-methyl rather thanphosphodiesterase linkages within the oligonucleotide backbone.

As used herein, the term “an agent that sensitizes tumor cells to theeffects of EGFR kinase inhibitors” when used without furtherqualification as to the nature of the agent, refers to an mTORinhibitor. An mTOR inhibitor can be any mTOR inhibitor that is currentlyknown in the art or that will be identified in the future, and includesany chemical entity that, upon administration to a patient, results ininhibition of mTOR in the patient. An mTOR inhibitor can inhibit mTOR byany biochemical mechanism, including competition at the ATP bindingsite, competition elsewhere at the catalytic site of mTOR kinase,non-competitive inhibition, irreversible inhibition (e.g. covalentprotein modification), or modulation of the interactions of otherprotein subunits or binding proteins with mTOR kinase in a way thatresults in inhibition of mTOR kinase activity (e.g. modulation of theinteraction of mTOR with FKBP12, GβL, (mLST8), RAPTOR (mKOG1), or RICTOR(mAVO3)). Specific examples of mTOR inhibitors include: rapamycin; otherrapamycin macrolides, or rapamycin analogues, derivatives or prodrugs;RAD001 (also known as Everolimus, RAD001 is an alkylated rapamycin(40-O-(2-hydroxyethyl)-rapamycin), disclosed in U.S. Pat. No. 5,665,772;Novartis); CCI-779 (also known as Temsirolimus, CCI-779 is an ester ofrapamycin (42-ester with 3-hydroxy-2-hydroxymethyl-2-methylpropionicacid), disclosed in U.S. Pat. No. 5,362,718; Wyeth); AP23573 or AP23841(Ariad Pharmaceuticals); ABT-578 (40-epi-(tetrazolyl)-rapamycin; AbbottLaboratories); KU-0059475 (Kudus Pharmaceuticals); and TAFA-93 (arapamycin prodrug; Isotechnika). Examples of rapamycin analogs andderivatives known in the art include those compounds described in U.S.Pat. Nos. 6,329,386; 6,200,985; 6,117,863; 6,015,815; 6,015,809;6,004,973; 5,985,890; 5,955,457; 5,922,730; 5,912,253; 5,780,462;5,665,772; 5,637,590; 5,567,709; 5,563,145; 5,559,122; 5,559,120;5,559,119; 5,559,112; 5,550,133; 5,541,192; 5,541,191; 5,532,355;5,530,121; 5,530,007; 5,525,610; 5,521,194; 5,519,031; 5,516,780;5,508,399; 5,508,290; 5,508,286; 5,508,285; 5,504,291; 5,504,204;5,491,231; 5,489,680; 5,489,595; 5,488,054; 5,486,524; 5,486,523;5,486,522; 5,484,791; 5,484,790; 5,480,989; 5,480,988; 5,463,048;5,446,048; 5,434,260; 5,411,967; 5,391,730; 5,389,639; 5,385,910;5,385,909; 5,385,908; 5,378,836; 5,378,696; 5,373,014; 5,362,718;5,358,944; 5,346,893; 5,344,833; 5,302,584; 5,262,424; 5,262,423;5,260,300; 5,260,299; 5,233,036; 5,221,740; 5,221,670; 5,202,332;5,194,447; 5,177,203; 5,169,851; 5,164,399; 5,162,333; 5,151,413;5,138,051; 5,130,307; 5,120,842; 5,120,727; 5,120,726; 5,120,725;5,118,678; 5,118,677; 5,100,883; 5,023,264; 5,023,263; and 5,023,262;all of which are incorporated herein by reference. Rapamycin derivativesare also disclosed for example in WO 94/09010, WO 95/16691, WO 96/41807,or WO 99/15530, which are incorporated herein by reference. Such analogsand derivatives include 32-deoxorapamycin,16-pent-2-ynyloxy-32-deoxorapamycin, 16-pent-2-ynyloxy-32 (S orR)-dihydro-rapamycin, 16-pent-2-ynyloxy-32 (S orR)-dihydro-40-O-(2-hydroxyethyl)-rapamycin,40-0-(2-hydroxyethyl)-rapamycin, 32-deoxorapamycin and16-pent-2-ynyloxy-32(S)-dihydro-rapamycin. Rapamycin derivatives mayalso include the so-called rapalogs, e.g. as disclosed in WO 98/02441and WO01/14387 (e.g. AP23573, AP23464, AP23675 or AP23841). Furtherexamples of a rapamycin derivative are those disclosed under the namebiolimus-7 or biolimus-9 (BIOLIMUS A9™) (Biosensors International,Singapore). Any of the above rapamycin analogs or derivatives may bereadily prepared by procedures as described in the above references.

Additional examples of mTOR inhibitors useful in the invention describedherein include those disclosed and claimed in U.S. patent applicationSer. No. 11/599,663, filed Nov. 15, 2006, a series of compounds thatinhibit mTOR by binding to and directly inhibiting both mTORC1 andmTORC2 kinases. The latter application is incorporated herein in itsentirety. Examples of such compounds and their synthesis are describedherein in the Experimental Methods section below (under “Drugs”). Twosuch compounds are Compound A and Compound B, for which data indicatingtheir utility in the methods of this invention is included and describedherein. These two compounds exhibit either synergy or additivity ininhibiting tumor cell growth or proliferation when used in combinationwith an EGFR kinase inhibitor, depending on the tumor cell type and EMTstatus. Synergy is observed in the majority of tumor cell types (e.g.see Experimental Details herein). Similar results can be obtained withany compound that inhibits mTOR by binding to and directly inhibitingboth mTORC1 and mTORC2 kinases, such as whose structures are disclosedherein (see Experimental Section). Additional such compounds can readilybe identified by determining their ability to inhibit both mTORC1 andmTORC2 kinase activities using immunoprecipiation-kinase assays withantibodies specific to either the raptor or rictor proteins of themTORC1 and mTORC2 complexes (for an example of such assays, see Jacinto,E. et al. (2004) Nature Cell Biol. 6(11):1122-1128).

Compounds that inhibit mTOR by binding to and directly inhibiting bothmTORC1 and mTORC2 kinases have a number of important advantages overcompounds like rapamycin, or its analogues, that only directly inhibitmTORC1. These include (a) superior inhibition of pAkt and concomitantinduction of apoptosis in tumor cells, (b) complete inhibition of allphosphorylation of 4E-BP1, which results in greater anti-proliferativeeffects, (c) inhibition of pAkt (S473) in all tumor cells, thus leadingto superior pro-apoptotic effects (rapamycin inhibits pAkt (S473) inonly ˜20% of cancer cell lines), (d) treatment does not increase pAkt(S473) in any cancer cell type tested, and so does not promote tumorcell survival (unlike rapamycin treatment, which increases pAkt (S473)in ˜65% of cell lines) and (e) anti-proliferative activity in a farbroader spectrum of tumor cells (N.B. approximately 50% of cell lines ina given tumor type are insensitive to rapamycin).

Also useful in the invention described herein are mTOR inhibitors thatare dual PI3K/mTOR kinase inhibitors, such as for example the compoundPI-103 as described in Fan, Q-W et al (2006) Cancer Cell 9:341-349 andKnight, Z. A. et al. (2006) Cell 125:733-747.

Compounds that inhibit mTOR kinase, but are non-specific kinaseinhibitors that are relatively toxic to normal non-neoplastic cells andthus not suitable for administration as a therapeutic, such as forexample the PI3 kinase inhibitors wortmannin and LY294002 (Brunn G. J.et al (1996) Embo J. 15:5256-5267), are not suitable for use in themethods of the invention described herein.

The present invention also encompasses the use of a combination of anEGFR kinase inhibitor and an mTOR inhibitor, for the manufacture of amedicament for the treatment of NSCL, pancreatic, colon or breast cancertumors or tumor metastases in a patient in need thereof, wherein eachinhibitor in the combination can be administered to the patient eithersimultaneously or sequentially. The present invention also encompassesthe use of a synergistically effective combination of an EGFR kinaseinhibitor and an mTOR inhibitor, for the manufacture of a medicament forthe treatment of NSCL, pancreatic, colon or breast cancer tumors ortumor metastases in a patient in need thereof, wherein each inhibitor inthe combination can be administered to the patient either simultaneouslyor sequentially. The present invention also encompasses the use of acombination of an EGFR kinase inhibitor and an agent that sensitizestumor cells to the effects of EGFR kinase inhibitors, wherein said agentis an mTOR inhibitor, for the manufacture of a medicament for thetreatment of NSCL, pancreatic, colon or breast cancer tumors or tumormetastases in a patient in need thereof, wherein each inhibitor in thecombination can be administered to the patient either simultaneously orsequentially. In an embodiment of any of the above uses, the cells ofthe NSCL, pancreatic, colon or breast cancer tumors or tumor metastaseshave high sensitivity or are very sensitive to growth inhibition by EGFRkinase inhibitors such as erlotinib as single agents (i.e. without anyagent that sensitizes the tumor cells to the effects of EGFR kinaseinhibitors), such as epithelial cells that have not undergone any formof EMT (e.g. like H292, H358, or BxPC3 tumor cells). In anotherembodiment of any of the above uses, the cells of the NSCL, pancreatic,colon or breast cancer tumors or tumor metastases have low sensitivityor are relatively insensitive to growth inhibition by EGFR kinaseinhibitors such as erlotinib as single agents, such as epithelial cellsthat have undergone an EMT and have acquired mesenchymal characteristics(e.g. like H460 or Calu6 tumor cells). In an alternative embodiment ofany of the above uses the present invention also encompasses the use ofan EGFR kinase inhibitor and mTOR inhibitor combination in combinationwith another anti-cancer agent or agent that enhances the effect of suchan agent for the manufacture of a medicament for the treatment of NSCL,pancreatic, colon or breast cancer tumors or tumor metastases in apatient in need thereof, wherein each inhibitor in the combination canbe administered to the patient either simultaneously or sequentially. Inthis context, the other anti-cancer agent or agent that enhances theeffect of such an agent can be any of the agents listed above that canbe added to the EGFR kinase inhibitor and mTOR inhibitor combinationwhen treating patients.

The invention also encompasses a pharmaceutical composition that iscomprised of a combination of an EGFR kinase inhibitor and an agent thatsensitizes tumor cells to the effects of EGFR kinase inhibitors incombination with a pharmaceutically acceptable carrier.

Preferably the composition is comprised of a pharmaceutically acceptablecarrier and a non-toxic therapeutically effective amount of acombination of an EGFR kinase inhibitor and an agent that sensitizestumor cells to the effects of EGFR kinase inhibitors (includingpharmaceutically acceptable salts of each component thereof).

Moreover, within this preferred embodiment, the invention encompasses apharmaceutical composition for the treatment of disease, the use ofwhich results in the inhibition of growth of neoplastic cells, benign ormalignant tumors, or metastases, comprising a pharmaceuticallyacceptable carrier and a non-toxic therapeutically effective amount of acombination of an EGFR kinase inhibitor and an agent that sensitizestumor cells to the effects of EGFR kinase inhibitors (includingpharmaceutically acceptable salts of each component thereof).

The term “pharmaceutically acceptable salts” refers to salts preparedfrom pharmaceutically acceptable non-toxic bases or acids. When acompound of the present invention is acidic, its corresponding salt canbe conveniently prepared from pharmaceutically acceptable non-toxicbases, including inorganic bases and organic bases. Salts derived fromsuch inorganic bases include aluminum, ammonium, calcium, copper (cupricand cuprous), ferric, ferrous, lithium, magnesium, manganese (manganicand manganous), potassium, sodium, zinc and the like salts. Particularlypreferred are the ammonium, calcium, magnesium, potassium and sodiumsalts. Salts derived from pharmaceutically acceptable organic non-toxicbases include salts of primary, secondary, and tertiary amines, as wellas cyclic amines and substituted amines such as naturally occurring andsynthesized substituted amines. Other pharmaceutically acceptableorganic non-toxic bases from which salts can be formed include ionexchange resins such as, for example, arginine, betaine, caffeine,choline, N′,N′-dibenzylethylenediamine, diethylamine,2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine,ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, glucamine,glucosamine, histidine, hydrabamine, isopropylamine, lysine,methylglucamine, morpholine, piperazine, piperidine, polyamine resins,procaine, purines, theobromine, triethylameine, trimethylamine,tripropylamine, tromethamine and the like.

When a compound of the present invention is basic, its correspondingsalt can be conveniently prepared from pharmaceutically acceptablenon-toxic acids, including inorganic and organic acids. Such acidsinclude, for example, acetic, benzenesulfonic, benzoic, camphorsulfonic,citric, ethanesulfonic, fumaric, gluconic, glutamic, hydrobromic,hydrochloric, isethionic, lactic, maleic, malic, mandelic,methanesulfonic, mucic, nitric, pamoic, pantothenic, phosphoric,succinic, sulfuric, tartaric, p-toluenesulfonic acid and the like.Particularly preferred are citric, hydrobromic, hydrochloric, maleic,phosphoric, sulfuric and tartaric acids.

The pharmaceutical compositions of the present invention comprise acombination of an EGFR kinase inhibitor and an agent that sensitizestumor cells to the effects of EGFR kinase inhibitors (includingpharmaceutically acceptable salts of each component thereof) as activeingredients, a pharmaceutically acceptable carrier and optionally othertherapeutic ingredients or adjuvants. Other therapeutic agents mayinclude those cytotoxic, chemotherapeutic or anti-cancer agents, oragents which enhance the effects of such agents, as listed above. Thecompositions include compositions suitable for oral, rectal, topical,and parenteral (including subcutaneous, intramuscular, and intravenous)administration, although the most suitable route in any given case willdepend on the particular host, and nature and severity of the conditionsfor which the active ingredient is being administered. Thepharmaceutical compositions may be conveniently presented in unit dosageform and prepared by any of the methods well known in the art ofpharmacy.

In practice, the compounds represented by the combination of an EGFRkinase inhibitor and an agent that sensitizes tumor cells to the effectsof EGFR kinase inhibitors (including pharmaceutically acceptable saltsof each component thereof) of this invention can be combined as theactive ingredient in intimate admixture with a pharmaceutical carrieraccording to conventional pharmaceutical compounding techniques. Thecarrier may take a wide variety of forms depending on the form ofpreparation desired for administration, e.g. oral or parenteral(including intravenous). Thus, the pharmaceutical compositions of thepresent invention can be presented as discrete units suitable for oraladministration such as capsules, cachets or tablets each containing apredetermined amount of the active ingredient. Further, the compositionscan be presented as a powder, as granules, as a solution, as asuspension in an aqueous liquid, as a non-aqueous liquid, as anoil-in-water emulsion, or as a water-in-oil liquid emulsion. In additionto the common dosage forms set out above, a combination of an EGFRkinase inhibitor and an agent that sensitizes tumor cells to the effectsof EGFR kinase inhibitors (including pharmaceutically acceptable saltsof each component thereof) may also be administered by controlledrelease means and/or delivery devices. The combination compositions maybe prepared by any of the methods of pharmacy. In general, such methodsinclude a step of bringing into association the active ingredients withthe carrier that constitutes one or more necessary ingredients. Ingeneral, the compositions are prepared by uniformly and intimatelyadmixing the active ingredient with liquid carriers or finely dividedsolid carriers or both. The product can then be conveniently shaped intothe desired presentation.

Thus, the pharmaceutical compositions of this invention may include apharmaceutically acceptable carrier and a combination of an EGFR kinaseinhibitor and an agent that sensitizes tumor cells to the effects ofEGFR kinase inhibitors (including pharmaceutically acceptable salts ofeach component thereof). A combination of an EGFR kinase inhibitor andan agent that sensitizes tumor cells to the effects of EGFR kinaseinhibitors (including pharmaceutically acceptable salts of eachcomponent thereof), can also be included in pharmaceutical compositionsin combination with one or more other therapeutically active compounds.Other therapeutically active compounds may include those cytotoxic,chemotherapeutic or anti-cancer agents, or agents which enhance theeffects of such agents, as listed above.

Thus in one embodiment of this invention, a pharmaceutical compositioncan comprise a combination of an EGFR kinase inhibitor and an agent thatsensitizes tumor cells to the effects of EGFR kinase inhibitors incombination with an anticancer agent, wherein said anti-cancer agent isa member selected from the group consisting of alkylating drugs,antimetabolites, microtubule inhibitors, podophyllotoxins, antibiotics,nitrosoureas, hormone therapies, kinase inhibitors, activators of tumorcell apoptosis, and antiangiogenic agents.

The pharmaceutical carrier employed can be, for example, a solid,liquid, or gas. Examples of solid carriers include lactose, terra alba,sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate, andstearic acid. Examples of liquid carriers are sugar syrup, peanut oil,olive oil, and water. Examples of gaseous carriers include carbondioxide and nitrogen.

In preparing the compositions for oral dosage form, any convenientpharmaceutical media may be employed. For example, water, glycols, oils,alcohols, flavoring agents, preservatives, coloring agents, and the likemay be used to form oral liquid preparations such as suspensions,elixirs and solutions; while carriers such as starches, sugars,microcrystalline cellulose, diluents, granulating agents, lubricants,binders, disintegrating agents, and the like may be used to form oralsolid preparations such as powders, capsules and tablets. Because oftheir ease of administration, tablets and capsules are the preferredoral dosage units whereby solid pharmaceutical carriers are employed.Optionally, tablets may be coated by standard aqueous or nonaqueoustechniques.

A tablet containing the composition of this invention may be prepared bycompression or molding, optionally with one or more accessoryingredients or adjuvants. Compressed tablets may be prepared bycompressing, in a suitable machine, the active ingredient in afree-flowing form such as powder or granules, optionally mixed with abinder, lubricant, inert diluent, surface active or dispersing agent.Molded tablets may be made by molding in a suitable machine, a mixtureof the powdered compound moistened with an inert liquid diluent. Eachtablet preferably contains from about 0.05 mg to about 5 g of the activeingredient and each cachet or capsule preferably contains from about0.05 mg to about 5 g of the active ingredient.

For example, a formulation intended for the oral administration tohumans may contain from about 0.5 mg to about 5 g of active agent,compounded with an appropriate and convenient amount of carrier materialthat may vary from about 5 to about 95 percent of the total composition.Unit dosage forms will generally contain between from about 1 mg toabout 2 g of the active ingredient, typically 25 mg, 50 mg, 100 mg, 200mg, 300 mg, 400 mg, 500 mg, 600 mg, 800 mg, or 1000 mg.

Pharmaceutical compositions of the present invention suitable forparenteral administration may be prepared as solutions or suspensions ofthe active compounds in water. A suitable surfactant can be includedsuch as, for example, hydroxypropylcellulose. Dispersions can also beprepared in glycerol, liquid polyethylene glycols, and mixtures thereofin oils. Further, a preservative can be included to prevent thedetrimental growth of microorganisms.

Pharmaceutical compositions of the present invention suitable forinjectable use include sterile aqueous solutions or dispersions.Furthermore, the compositions can be in the form of sterile powders forthe extemporaneous preparation of such sterile injectable solutions ordispersions. In all cases, the final injectable form must be sterile andmust be effectively fluid for easy syringability. The pharmaceuticalcompositions must be stable under the conditions of manufacture andstorage; thus, preferably should be preserved against the contaminatingaction of microorganisms such as bacteria and fungi. The carrier can bea solvent or dispersion medium containing, for example, water, ethanol,polyol (e.g., glycerol, propylene glycol and liquid polyethyleneglycol), vegetable oils, and suitable mixtures thereof.

Pharmaceutical compositions of the present invention can be in a formsuitable for topical sue such as, for example, an aerosol, cream,ointment, lotion, dusting powder, or the like. Further, the compositionscan be in a form suitable for use in transdermal devices. Theseformulations may be prepared, utilizing a combination of an EGFR kinaseinhibitor and an agent that sensitizes tumor cells to the effects ofEGFR kinase inhibitors (including pharmaceutically acceptable salts ofeach component thereof) of this invention, via conventional processingmethods. As an example, a cream or ointment is prepared by admixinghydrophilic material and water, together with about 5 wt % to about 10wt % of the compound, to produce a cream or ointment having a desiredconsistency.

Pharmaceutical compositions of this invention can be in a form suitablefor rectal administration wherein the carrier is a solid. It ispreferable that the mixture forms unit dose suppositories. Suitablecarriers include cocoa butter and other materials commonly used in theart. The suppositories may be conveniently formed by first admixing thecomposition with the softened or melted carrier(s) followed by chillingand shaping in molds.

In addition to the aforementioned carrier ingredients, thepharmaceutical formulations described above may include, as appropriate,one or more additional carrier ingredients such as diluents, buffers,flavoring agents, binders, surface-active agents, thickeners,lubricants, preservatives (including anti-oxidants) and the like.Furthermore, other adjuvants can be included to render the formulationisotonic with the blood of the intended recipient. Compositionscontaining a combination of an EGFR kinase inhibitor and an agent thatsensitizes tumor cells to the effects of EGFR kinase inhibitors(including pharmaceutically acceptable salts of each component thereof)may also be prepared in powder or liquid concentrate form.

Dosage levels for the compounds of the combination of this inventionwill be approximately as described herein, or as described in the artfor these compounds. It is understood, however, that the specific doselevel for any particular patient will depend upon a variety of factorsincluding the age, body weight, general health, sex, diet, time ofadministration, route of administration, rate of excretion, drugcombination and the severity of the particular disease undergoingtherapy.

The present invention also provides a method for treating tumors ortumor metastases in a patient, comprising administering to said patientsimultaneously or sequentially a therapeutically effective amount of acombination of an EGFR kinase inhibitor and an agent that sensitizestumor cells to the effects of EGFR kinase inhibitors, wherein said agentis an mTOR inhibitor that binds to and directly inhibits both mTORC1 andmTORC2 kinases. In one embodiment of this method the patient is a humanthat is being treated for cancer. In one embodiment of this method thecells of the tumors or tumor metastases are relatively insensitive orrefractory to treatment with an EGFR inhibitor as a single agent. In oneembodiment of this method the EGFR kinase inhibitor and mTOR inhibitorare co-administered to the patient in the same formulation. In anotherembodiment of this method the EGFR kinase inhibitor and mTOR inhibitorare co-administered to the patient in different formulations. In anotherembodiment of this method the EGFR kinase inhibitor and mTOR inhibitorare co-administered to the patient by the same route. In anotherembodiment of this method the EGFR kinase inhibitor and mTOR inhibitorare co-administered to the patient by different routes. In anotherembodiment of this method the EGFR kinase inhibitor is a small organicmolecule, an antibody or an antibody fragment that binds specifically tothe EGFR. In another embodiment of this method the EGFR kinase inhibitorcomprises erlotinib, or a salt thereof. In another embodiment of thismethod, one or more other anti-cancer agents may additionally beadministered to said patient. In another embodiment of this method theadministering to the patient is simultaneous. In another embodiment ofthis method the administering to the patient is sequential. In anotherembodiment of this method the cells of the tumors or tumor metastaseshave high sensitivity to growth inhibition by EGFR kinase inhibitors assingle agents. In another embodiment of this method the cells of thetumors or tumor metastases have low sensitivity to growth inhibition byEGFR kinase inhibitors as single agents. In another embodiment of thismethod the cells of the tumors or tumor metastases have not undergoneany form of EMT (e.g. epithelial cells). In another embodiment of thismethod the cells of the tumors or tumor metastases have undergone an EMT(i.e. mesenchymal or mesenchymal-like cells).

The present invention also provides a method for the treatment ofcancer, comprising administering to a subject in need of such treatmentan amount of the EGFR kinase inhibitor, or a pharmaceutically acceptablesalt thereof; and an amount of an mTOR inhibitor that binds to anddirectly inhibits both mTORC1 and mTORC2 kinases, or a pharmaceuticallyacceptable salt thereof; wherein at least one of the amounts isadministered as a sub-therapeutic amount. In one embodiment of thismethod the EGFR kinase inhibitor comprises erlotinib, or a salt thereof.In another embodiment of this method, one or more other anti-canceragents may additionally be administered to said patient. In oneembodiment of this method the cancer is relatively insensitive orrefractory to treatment with an EGFR inhibitor as a single agent.

The present invention also provides a method for treating tumors ortumor metastases in a patient, comprising administering to said patientsimultaneously or sequentially a synergistically effective therapeuticamount of a combination of an EGFR kinase inhibitor and an mTORinhibitor that binds to and directly inhibits both mTORC1 and mTORC2kinases. In one embodiment of this method the EGFR kinase inhibitorcomprises erlotinib, or a salt thereof. In another embodiment of thismethod, one or more other anti-cancer agents may additionally beadministered to said patient.

In one embodiment of this method the tumors or tumor metastases arerelatively insensitive or refractory to treatment with an EGFR inhibitoras a single agent.

The present invention also provides a method for treating tumors ortumor metastases in a patient, comprising the steps of diagnosing apatient's likely responsiveness to an EGFR kinase inhibitor by assessingwhether the tumor cells have undergone an epithelial-mesenchymaltransition, identifying the patient as one whose tumor or tumormetastases cells have undergone an epithelial-mesenchymal transition andare thus predicted to be relatively insensitive to an EGFR kinaseinhibitor as a single agent, and thus likely to show an enhancedresponse in the presence of an mTOR inhibitor, and administering to saidpatient simultaneously or sequentially a therapeutically effectiveamount of a combination of an EGFR kinase inhibitor and an mTORinhibitor that binds to and directly inhibits both mTORC1 and mTORC2kinases.

The present invention also provides a method for treating tumors ortumor metastases in a patient refractory to treatment with an EGFRkinase inhibitor as a single agent, comprising administering to saidpatient simultaneously or sequentially a therapeutically effectiveamount of a combination of an EGFR kinase inhibitor and an mTORinhibitor that binds to and directly inhibits both mTORC1 and mTORC2kinases.

The present invention also provides a pharmaceutical compositioncomprising an EGFR kinase inhibitor and an mTOR inhibitor that binds toand directly inhibits both mTORC1 and mTORC2 kinases, in apharmaceutically acceptable carrier. In one embodiment of thepharmaceutical composition the EGFR kinase inhibitor compriseserlotinib. In one embodiment of the pharmaceutical composition theerlotinib in the composition is present as a hydrochloride salt. In oneembodiment the pharmaceutical composition additionally comprises one ormore other anti-cancer agents.

The present invention also provides a kit comprising a container,comprising an mTOR inhibitor that binds to and directly inhibits bothmTORC1 and mTORC2 kinases, and an EGFR kinase inhibitor. In oneembodiment of the kit the EGFR kinase inhibitor comprises erlotinib. Inone embodiment the kit further comprises a sterile diluent. In oneembodiment the kit further comprises a package insert comprising printedinstructions directing the use of a combined treatment of an mTORinhibitor that binds to and directly inhibits both mTORC1 and mTORC2kinases and erlotinib to a patient as a method for treating tumors,tumor metastases or other cancers in a patient.

In the preceding methods of treatment of a patient or subject using anmTOR inhibitor that binds to and directly inhibits both mTORC1 andmTORC2 kinases, the patient or subject is preferably a human in need oftreatment for cancer, or a precancerous condition or lesion, selectedfrom the list of such conditions provided herein above. Cancersparticularly suitable for these methods of treatment include for exampleNSCLC and pancreatic cancer, especially mesenchymal or late stagecancers of these types where a synergistic outcome is obtained, and alsoovarian cancer, head and neck squamous cell carcinoma and breast cancer,in all of which a synergistic effect is also frequently observed.

In further embodiments of any of the above methods, compositions or kitsof this invention where an mTOR inhibitor that binds to and directlyinhibits both mTORC1 and mTORC2 kinases is used, the mTOR inhibitorcomprises a compound of Formula (I) as described herein.

This invention will be better understood from the Experimental Detailsthat follow. However, one skilled in the art will readily appreciatethat the specific methods and results discussed are merely illustrativeof the invention as described more fully in the claims which followthereafter, and are not to be considered in any way limited thereto.

EXPERIMENTAL DETAILS

Recent reports in the literature have suggested that combining EGFRinhibitors with agents that antagonize downstream signaling pathways maypermit sensitization in cell lines that either have redundancy inreceptor tyrosine kinase signaling or contain specific mutations indownstream signaling. Herein, the present inventors have determined thecorrelation between erlotinib's ability to regulate the activity of thePI3K-PDK1-Akt-mTOR pathway and sensitivity to growth inhibition in agroup of 22 cell lines derived from breast, colon, pancreatic and NSCLtumors. It was found that in cell lines sensitive to growth inhibitionby erlotinib there is down-modulation of the activity of this pathway.In less sensitive cell lines, erlotinib is ineffective at fullydown-regulating S6 ribosomal protein (below basal levels), a substratedownstream of mTOR. Here mTOR activity is likely controlled, at least tosome extent, by EGFR independent mechanisms, including other growthfactors or mutations.

It had not been previously determined if it was possible to combineerlotinib with another targeted agent in order to sensitize breast,colon, pancreatic or NSCL tumor cells that poorly respond to erlotinibas a single agent. Unlike cytotoxic chemotherapies that often sharesimilar toxicities, molecular targeted agents tend to havenon-overlapping toxicities and thus identifying cocktails of targetedagents to block cancer cell growth may be more clinically feasible. Thepresent inventors have determined the effect of combining erlotinib withrapamycin, a targeted agent that acts downstream of EGFR to directlyinactivate mTOR. Rapamycin is a high molecular weight polyketide naturalproduct derived from a soil bacteria identified on the island Rapa Nui.It acts by disrupting the protein-protein interactions between raptorand mTOR. mTOR requires raptor to interact with a number of substrateproteins, including 4EBP1 and S6K, thus inhibiting this interactionblocks some of mTOR's functions. Synergistic behavior of the mTORinhibitor rapamycin combined with EGFR inhibitors to block tumor cellgrowth had been previously reported for renal cell carcinoma andglioblastomas, but had not been examined in breast, colon, pancreatic orNSCL tumors.

Herein, it is demonstrated that rapamycin can re-sensitize breast,colon, pancreatic or NSCL tumor cell lines that are relativelyinsensitive to erlotinib as a single agent. Thus combining an mTORinhibitor with an EGFR kinase inhibitor such as erlotinib should beuseful clinically in patients with breast, colon, pancreatic or NSCLtumors.

Materials and Methods

Drugs.

The selective HER1/EGFR kinase inhibitor, erlotinib, was synthesized byOSI Pharmaceuticals, Uniondale, N.Y., USA, as the hydrochloride salt,erlotinib HCl (TARCEVA®).

Rapamycin, for in vitro experiments, was purchased from Sigma AldrichChemicals (St. Louis, Mo.), and for xenograft experiments, from LCLaboratories (Woburn, Mass.).

Examples of mTOR kinase inhibitors that inhibit mTOR by binding to anddirectly inhibiting both mTORC1 and mTORC2 kinases include compoundsrepresented by Formula (I) as described below. Compounds A and Brepresent mTOR inhibitors according to Formula (I).

or a pharmaceutically acceptable salt thereof, wherein:

X₁, and X₂ are each independently N or C-(E¹)_(aa);

X₅ is N, C-(E¹)_(aa), or N-(E)_(aa);

X₃, X₄, X₆, and X₇ are each independently N or C;

wherein at least one of X₃, X₄, X₅, X₆, and X₇ is independently N orN-(E¹)_(aa);

R³ is C₀₋₁₀alkyl, cycloC₃₋₁₀alkyl, aminomethylcycloC₃₋₁₀alkyl,bicycloC₅₋₁₀alkyl, aryl, heteroaryl, aralkyl, heteroaralkyl,heterocyclyl or heterobicycloC₅₋₁₀alkyl any of which is optionallysubstituted by one or more independent G¹¹ substituents;

Q¹ is -A(R¹)_(m)B(W)_(n) or —B(G¹¹)_(n)A(Y)_(m);

A and B are respectively, 5 and 6 membered aromatic or heteroaromaticrings, fused together to form a 9-membered heteroaromatic systemexcluding 5-benzo[b]furyl and 3-indolyl; and excluding 2-indolyl,2-benzoxazole, 2-benzothiazole, 2-benzimidazolyl,4-aminopyrrolopyrimidin-5-yl, 4-aminopyrrolopyrimidin-6-yl, and7-deaza-7-adenosinyl derivatives when X₁ and X₅ are CH, X₃, X₆ and X₇are C, and X₂ and X₄ are N;

or Q¹ is -A(R¹)_(m)A(Y)_(m), wherein each A is the same or different5-membered aromatic or heteroaromatic ring, and the two are fusedtogether to form an 8-membered heteroaromatic system;

R¹ is independently, hydrogen, —N(C₀₋₈alkyl)(C₀₋₈alkyl), hydroxyl,halogen, oxo, aryl (optionally substituted with 1 or more R³¹ groups),hetaryl (optionally substituted with 1 or more R³¹ groups), C₁₋₆alkyl,—C₀₋₈alkylC₃₋₈cycloalkyl, —C₀₋₈alkyl-NR³¹¹S(O)₀₋₂R³²¹,—C₀₋₈alkyl-NR³¹¹S(O)₀₋₂NR³²¹R³³¹, —C₀₋₈alkyl-S(O)₀₋₂NR³¹¹R³²¹,—C₀₋₈alkyl-NR³¹¹COR³²¹,—C₀₋₈alkyl-NR³¹¹CO₂R³²¹—C₀₋₈alkyl-NR³¹¹CONR³²¹R³³¹,—C₀₋₈-alkyl-CONR³¹¹R³²¹, —C₀₋₈alkyl-CON(R³¹¹)S(O)₀₋₂R³²¹,—C₀₋₈alkyl-CO₂R³¹¹, —C₀₋₈alkyl-S(O)₀₋₂R³¹¹, —C₀₋₈alkyl-O—C₁₋₈alkyl,—C₀₋₈alkyl-O—C₀₋₈alkylC₃₋₈cycloalkyl,—C₀₋₈alkyl-O—C₀₋₈alkylheterocyclyl, —C₀₋₈alkyl-O—C₀₋₈alkylaryl,—C₀₋₈alkylaryl, —C₀₋₈alkylhetaryl, —C₀₋₈alkylheterocyclyl,—C₀₋₈alkyl-O—C₀₋₈alkylhetaryl, —C₀₋₈alkyl-S—C₀₋₈alkyl,—C₀₋₈alkyl-S—C₀₋₈alkylC₃₋₈cycloalkyl,—C₀₋₈alkyl-S—C₀₋₈alkylheterocyclyl, —C₀₋₈alkyl-S—C₀₋₈alkylaryl,—C₀₋₈alkyl-S—C₀₋₈alkylhetaryl, —C₀₋₈alkyl-N(R³¹¹)—C₀₋₈alkyl,—C₀₋₈alkyl-N(R³¹¹)—C₀₋₈alkylC₃₋₈cycloalkyl,—C₀₋₈alkyl-N(R³¹¹)—C₀₋₈alkylheterocyclyl,—C₀₋₈alkyl-N(R¹¹)—C₀₋₈alkylaryl, —C₀₋₈alkyl-N(R³¹¹)—C₀₋₈alkylhetaryl,—C₀₋₈alkyl-NR³¹¹R³²¹, —C₂₋₈alkenyl, —C₂₋₈alkynyl, NO₂, CN, CF₃, OCF₃,OCHF₂; provided that Q¹ is not N-methyl-2-indolyl,N-(phenylsulfonyl)-2-indolyl, or N-tert-butoxycarbonyl

W is independently, hydrogen, —N(C₀₋₈alkyl)(C₀₋₈alkyl), hydroxyl,halogen, oxo, aryl (optionally substituted with 1 or more R³¹ groups),hetaryl (optionally substituted with 1 or more R³¹ groups), C₁₋₆alkyl,—C₀₋₈alkylC₃₋₈cycloalkyl, —C₀₋₈alkyl-NR³¹²S(O)₀₋₂R³²²,—C₀₋₈alkyl-NR³¹¹S(O)⁰-2NR³²¹R³³¹, —C₀₋₈alkyl-NR³¹¹CO₂R³²¹,—C₀₋₈alkyl-CON(R³¹¹)S(O)₀₋₂R³²¹, —C₀₋₈alkyl-S(O)₀₋₂NR³¹²R³²²,—C₀₋₈alkyl-NR³¹²COR³²², —C₀₋₈alkyl-NR³¹²CONR³²²R³³²,—C₀₋₈alkyl-CONR³¹²R³²², —C₀₋₈alkyl-CO₂R³¹², —C₀₋₈-alkylS(O)₀₋₂R³¹²,—C₀₋₈alkyl-O—C₁₋₈alkyl, —C₀₋₈alkyl-O—C₀₋₈alkylcyclyl,—C₀₋₈alkyl-O—C₀₋₈alkylheterocycloalkyl, —C₀₋₈alkyl-O—C₀₋₈alkylaryl,—Oaryl, —C₀₋₈alkyl-O—C₀₋₈alkylhetaryl, —C₀₋₈alkylaryl,—C₀₋₈alkylhetaryl, —C₀₋₈alkylheterocyclyl, —C₀₋₈alkyl-S—C₀₋₈alkyl,—C₀₋₈alkyl-S—C₀₋₈alkylC₃₋₈cycloalkyl,—C₀₋₈alkyl-S—C₀₋₈alkylheterocycloalkyl, —C₀₋₈alkyl-S—C₀₋₈alkylaryl,—C₀₋₈alkyl-S—C₀₋₈alkylhetaryl, —C₀₋₈alkyl-N(R³¹²)—C₀₋₈alkyl,—C₀₋₈alkyl-N(R³¹²)—C₀₋₈alkylC₃₋₈cycloalkyl,—C₀₋₈alkyl-N(R³¹²)—C₀₋₈alkylheterocycloalkyl,—C₀₋₈alkyl-N(R³¹²)—C₀₋₈alkylaryl, —C₀₋₈alkyl-N(R³¹²)—C₀₋₈alkylhetaryl,—C₀₋₈alkyl-NR³¹²R³²², —C₂₋₈alkenyl, —C₂₋₈alkynyl, NO₂, CN, CF₃, OCF₃,OCHF₂; provided that Q¹ is not 4-benzyloxy-2-indolyl;

Y is independently, hydrogen, —N(C₀₋₈alkyl)(C₀₋₈alkyl), hydroxyl,halogen, oxo, aryl (optionally substituted with 1 or more R³¹ groups),hetaryl (optionally substituted with 1 or more R³¹ groups), C₀₋₆alkyl,—C₀₋₈alkylC₃₋₈cycloalkyl, —C₀₋₈alkyl-NR³¹¹S(O)₀₋₂R³²¹,—C₀₋₈alkyl-NR³¹¹S(O)₀₋₂NR³²¹R³³¹, —C₀₋₈alkyl-NR³¹¹CO₂R³²¹,—C₀₋₈alkyl-CON(R³¹¹)S(O)₀₋₂R³²¹, —C₀₋₈alkyl-S(O)₀₋₂NR³¹¹R³²¹,—C₀₋₈alkyl-NR³¹¹COR³²¹, —C₀₋₈alkyl-NR³¹¹CONR³²¹R³³¹,—C₀₋₈alkyl-CONR³¹¹R³²¹, —C₀₋₈alkyl-CO₂R³¹¹, —C₀₋₈alkylS(O)₀₋₂R³¹¹,—C₀₋₈alkyl-O—C₁₋₈alkyl, —C₀₋₈alkyl-O—C₀₋₈alkylC₃₋₈cycloalkyl,—C₀₋₈alkyl-O—C₀₋₈alkylheterocycloalkyl, —C₀₋₈alkyl-O—C₀₋₈alkylaryl,—C₀₋₈alkyl-O—C₀₋₈alkylhetaryl, —C₀₋₈alkylaryl, —C₀₋₈alkylhetaryl,—C₀₋₈alkylheterocyclyl, —C₀₋₈alkyl-S—C₀₋₈alkyl,—C₀₋₈alkyl-S—C₀₋₈alkylC₃₋₈cycloalkyl,—C₀₋₈alkyl-S—C₀₋₈alkylheterocycloalkyl, —C₀₋₈alkyl-S—C₀₋₈alkylaryl,—C₀₋₈alkyl-S—C₀₋₈alkylhetaryl, —C₀₋₈alkyl-N(R³¹¹)—C₀₋₈alkyl,—C₀₋₈alkyl-N(R³¹¹)—C₀₋₈alkylC₃₋₈cycloalkyl,—C₀₋₈alkyl-N(R³¹¹)—C₀₋₈alkylheterocycloalkyl,—C₀₋₈alkyl-N(R³¹¹)—C₀₋₈alkylaryl, —C₀₋₈alkyl-N(R³)—C₀₋₈alkylhetaryl,—C₀₋₈alkyl-NR³¹¹R³²¹, —C₂₋₈alkenyl, —C₂₋₈alkynyl, NO₂, CN, CF₃, OCF₃,OCHF₂; provided that Q¹ is not 2-carboxy-5-benzo[b]thiophenyl;

G¹¹ is halo, oxo, —CF₃, —OCF₃, —OR³¹², —NR³¹²R³²², —C(O)R³¹²,—C(O)C₃₋₈cycloalkyl, —CO₂C₃₋₈cycloalkyl, —CO₂R³¹², —C(═O)NR³¹²R³²²,—NO₂, —CN, —S(O)₀₋₂R³¹², —SO₂NR³¹²R³²², NR³¹²(C═O)R³²², NR³¹²C(═O)OR³²²,NR³¹²C(═O)NR³²²R³³², NR³¹²S(O)₀₋₂R³²², —C(═S)OR³¹², —C(═O)SR³¹²,—NR³¹²C(═NR³²²)NR³³²R³⁴¹, —NR³¹²C(═NR³²²)OR³³², —NR³¹²C(═NR³²²)SR³³²,—OC(═O)OR³¹², —OC(═O)NR³¹²R³²², —OC(═O)SR³¹², —SC(═O)OR³¹²,—SC(═O)NR³¹²R³²², —P(O)OR³¹²OR³²², C₁₋₁₀alkylidene, C₀₋₁₀alkyl,C₂₋₁₀alkenyl, C₂₋₁₀alkynyl, —C₁₋₁₀alkoxyC₀₋₁₀alkyl,—C₁₋₁₀alkoxyC₂₋₁₀alkenyl, —C₁₋₁₀alkoxyC₂₋₁₀alkynyl,—C₁₋₁₀alkylthioC₁₋₁₀alkyl, —C₁₋₁₀alkylthioC₂₋₁₀alkenyl,—C₁₋₁₀alkylthioC₂₋₁₀alkynyl, cycloC₃₋₈alkyl, cycloC₃₋₈alkenyl,-cycloC₃₋₈alkylC₁₋₁₀alkyl, -cycloC₃₋₈alkenylC₁₋₁₀alkyl,-cycloC₃₋₈alkylC₂₋₁₀alkenyl, -cycloC₃₋₈alkenylC₂₋₁₀alkenyl,-cycloC₃₋₈alkylC₂₋₁₀alkynyl, -cycloC₃₋₈alkenylC₂₋₁₀alkynyl,-heterocyclyl-C₀₋₁₀alkyl, heterocyclyl-C₂₋₁₀alkenyl, or-heterocyclyl-C₂₋₁₀alkynyl, any of which is optionally substituted withone or more independent halo, oxo, —CF₃, —OCF₃, —OR³¹³, —NR³¹³R³²³,—C(O)R³¹³, —CO₂R³¹³, —C(═O)NR³¹³R³²³, —NO₂, —CN, —S(O)₀₋₂R³¹³,—SO₂NR³¹³R³²³, —NR³¹³C(═O)R³²³, —NR³¹³C(═O)OR³²³, —NR³¹³C(═O)NR³²³R³³³,—NR³¹³S(O)₀₋₂R³²³, —C(═S)OR³¹³, —C(═O)SR³¹³, —NR³¹³C(═NR³²³)NR³³³R³⁴²,—NR³¹³C(═NR³²³)OR³³³, —NR³¹³C(═NR³²³)SR³³³, —OC(═O)OR³³³,—OC(═O)NR³¹³R³²³, —OC(═O)SR³¹³, —SC(═O)OR³¹³, —P(O)OR³¹³⁰R³²³, or—SC(═O)NR³¹³R³²³ substituents;

or G¹¹ is aryl-C₀₋₁₀alkyl, aryl-C₂₋₁₀alkenyl, aryl-C₂₋₁₀alkynyl,hetaryl-C₀₋₁₀alkyl, hetaryl-C₂₋₁₀alkenyl, or hetaryl-C₂₋₁₀alkynyl, wherethe attachment point is from either the left or right as written, whereany of which is optionally substituted with one or more independenthalo, —CF₃, —OCF₃, —OR³¹³, —NR³¹³R³²³, —C(O)R³¹³, —CO₂R³¹³,—C(═O)NR³¹³R³²³, —NO₂, —CN, —S(O)₀₋₂R³¹³, —SO₂NR³¹³R³²³, NR³¹³C(═O)R³²³,—NR³¹³C(═O)OR³²³, —NR³¹³C(═O)NR³²³R³³³, —NR³¹³S(O)₀₋₂R³²³, —C(═S)OR³¹³,—C(═O)SR³¹³, —NR³²³C(═NR³¹³)NR³³³R³⁴², —NR³¹³C(═NR³²³)OR³³³,—NR³¹³C(═NR³²³)SR³³³, —OC(═O)OR³¹³, —OC(═O)NR³¹³R³²³, —OC(═O)SR³¹³,—SC(═O)OR³¹³, —P(O)OR³¹³OR³²³, or —SC(═O)NR³¹³R³²³ substituents;provided that G¹¹ is not N—CH₂CO₂H when R³ is 4-piperidinyl;

R³¹, R³², R³³, R³¹¹, R³²¹, R³³¹, R³¹², R³²², R³³², R³⁴¹, R³¹³, R³²³,R³³³, and R³⁴², in each instance, is independently

-   -   C₀₋₈alkyl optionally substituted with an aryl, heterocyclyl or        hetaryl substituent, or C₀₋₈alkyl optionally substituted with        1-6 independent halo, —CON(C₀₋₈alkyl)(C₀₋₈alkyl),        —CO(C₀₋₈alkyl), —OC₀₋₈alkyl, —Oaryl, —Ohetaryl, —Oheterocyclyl,        —S(O)₀₋₂aryl, —S(O)₀₋₂hetaryl, —S(O)₀₋₂heterocyclyl,        —S(O)₀₋₂C₀₋₈alkyl, —N(C₀₋₈alkyl)(C₀₋₈alkyl),        —N(C₀₋₈alkyl)CON(C₀₋₈alkyl)(C₀₋₈alkyl),        —N(C₀₋₈alkyl)CO(C₁₋₈alkyl), —N(C₀₋₈alkyl)CO(C₃₋₈cycloalkyl),        —N(C₀₋₈alkyl)CO₂(C₁₋₈alkyl), —S(O)₁₋₂N(C₀₋₈alkyl)(C₀₋₈alkyl),        —NR¹¹S(O)₁₋₂(C₀₋₈alkyl), —CON(C₃₋₈cycloalkyl)(C₃₋₈cycloalkyl),        —CON(C₀₋₈alkyl)(C₃₋₈cycloalkyl),        —N(C₃₋₈cycloalkyl)CON(C₀₋₈alkyl)(C₀₋₈alkyl),        —N(C₃₋₈cycloalkyl)CON(C₃₋₈cycloalkyl)(C₀₋₈alkyl),        —N(C₀₋₈alkyl)CON(C₃₋₈cycloalkyl)(C₀₋₈alkyl),        —N(C₀₋₈alkyl)CO₂(C₃₋₈cycloalkyl),        —N(C₃₋₈cycloalkyl)CO₂(C₃₋₈cycloalkyl),        S(O)₁₋₂N(C₀₋₈alkyl)(C₃₋₈cycloalkyl),        —NR¹¹S(O)₁₋₂(C₃₋₈cycloalkyl), C₂₋₈alkenyl, C₂₋₈alkynyl, CN, CF₃,        OH, or optionally substituted aryl substituents; such that each        of the above aryl, heterocyclyl, hetaryl, alkyl or cycloalkyl        groups may be optionally, independently substituted with        —N(C₀₋₈alkyl)(C₀₋₈alkyl), hydroxyl, halogen, oxo, aryl, hetaryl,        C₀₋₆alkyl, C₀₋₈alkylcyclyl,        —C₀₋₈alkyl-N(C₀₋₈alkyl)-S(O)₀₋₂—(C₀₋₈alkyl),        —C₀₋₈alkyl-S(O)₀₋₂—N(C₀₋₈alkyl)(C₀₋₈alkyl),        —C₀₋₈alkyl-N(C₀₋₈alkyl)CO(C₀₋₈alkyl),        —C₀₋₈alkyl-N(C₀₋₈alkyl)CO—N(C₀₋₈alkyl)(C₀₋₈alkyl),        —C₀₋₈alkyl-CO—N(C₀₋₈alkyl)(C₀₋₈alkyl),        —C₁₋₈alkyl-CO₂—(C₀₋₈alkyl), —C₀₋₈alkylS(O)₀₋₂—(C₀₋₈alkyl),        —C₀₋₈alkyl-O—C₁₋₈alkyl, —C₀₋₈alkyl-O—C₀₋₈alkylcyclyl,        —C₀₋₈alkyl-O—C₀₋₈alkylheterocyclyl, —C₀₋₈alkyl-O—C₀₋₈alkylaryl,        —C₀₋₈alkyl-O—C₀₋₈alkylhetaryl, —C₀₋₈alkyl-S—C₀₋₈alkyl,        —C₀₋₈alkyl-S—C₀₋₈alkylcyclyl,        —C₀₋₈alkyl-S—C₀₋₈alkylheterocyclyl, —C₀₋₈alkyl-S—C₀₋₈alkylaryl,        —C₀₋₈alkyl-S—C₀₋₈alkylhetaryl,        —C₀₋₈alkyl-N(C₀₋₈alkyl)-C₀₋₈alkyl,        —C₀₋₈alkyl-N(C₀₋₈alkyl)-C₀₋₈alkylcyclyl,        —C₀₋₈alkyl-N(C₀₋₈alkyl)-C₀₋₈alkylheterocyclyl,        —C₀₋₈alkyl-N(C₀₋₈alkyl)-C₀₋₈alkylaryl,        —C₀₋₈alkyl-N(C₀₋₈alkyl)-C₀₋₈alkylhetaryl, C₂₋₈alkenyl,        C₂₋₈alkynyl, NO₂, CN, CF₃, OCF₃, OCHF₂,        -   —C₀₋₈alkyl-C₃₋₈cycloalkyl,        -   —C₀₋₈alkyl-O—C₀₋₈alkyl,        -   —C₀₋₈alkyl-N(C₀₋₈alkyl)(C₀₋₈alkyl),        -   —C₀₋₈alkyl-S(O)₀₋₂—C₀₋₈alkyl, or    -   heterocyclyl optionally substituted with 1-4 independent        C₀₋₈alkyl, cyclyl, or substituted cyclyl substituents;

E in each instance is independently halo, —CF₃, —OCF₃, —OR², —NR³¹R³²,—C(═O)R³¹, —CO₂R³¹, —CONR³¹R³², —NO₂, —CN, —S(O)₀₋₂R³¹, —S(O)₀₋₂NR³¹R³²,—NR³¹C(═O)R³², —NR³¹C(═O)OR³², —NR³¹C(═O)NR³²R³³, —NR³¹¹S(O)₀₋₂R³²,—C(═S)OR³¹, —C(═O)SR³¹, —NR³¹C(═NR³²)NR³³R³¹, —NR³¹C(═NR³²)OR³³,—NR³¹C(═NR³¹¹)SR³¹, —OC(═O)O OC(═O)R³¹, —OC(═O)R³¹R³², —OC(═O)SR³¹,—SC(═O)OR³¹, —SC(═O)NR³¹R³², C₀₋₁₀alkyl, C₂₋₁₀alkenyl, C₂₋₁₀alkynyl,—C₁₋₁₀alkoxyC₁₋₁₀alkyl, —C₁₋₁₀alkoxyC₂₋₁₀alkenyl,—C₁₋₁₀alkoxyC₂₋₁₀alkynyl, —C₁₋₁₀alkylthioC₁₋₁₀alkyl,—C₁₋₁₀alkylthioC₂₋₁₀alkenyl, —C₁₋₁₀-alkylthioC₂₋₁₀alkynyl,cycloC₃₋₈alkyl, cycloC₃₋₈alkenyl, -cycloC₃₋₈alkylC₁₋₁₀alkyl,-cycloC₃₋₈alkenylC₁₋₁₀alkyl, -cycloC₃₋₈alkylC₂₋₁₀alkenyl,-cycloC₃₋₈alkenylC₂₋₁₀alkenyl, -cycloC₃₋₈alkylC₂₋₁₀alkynyl,-cycloC₃₋₈alkenylC₂₋₁₀alkynyl, -heterocyclyl-C₁₋₁₀alkyl,-heterocyclyl-C₂₋₁₀alkenyl, or -heterocyclyl-C₂₋₁₀alkynyl, any of whichis optionally substituted with one or more independent halo, oxo, —CF₃,—OCF₃, —OR³¹, —NR³¹R³², —C(═O)R³¹, —CO₂R³¹, —C(═O)NR³¹R³², —NO₂, —CN,—S(═O)₀₋₂R³¹, —SO₂NR³¹, —NR³¹C(═O)R³², —NR³¹C(═O)OR³¹,—NR³¹C(═O)NR³²R³³, —NR³¹¹S(O)₀₋₂R³¹, —C(═S)OR³¹, —C(═O)SR³¹,—NR³¹C(═NR³²)NR³³R³¹, —NR³¹C(═NR³²)OR³³, —NR³¹C(═NR³²)SR³³, —OC(═O)OR³¹,—OC(═O)NR³¹R³², —OC(═O)SR³¹, —SC(═O)OR³¹, or —SC(═O)NR³¹R³²substituents;

or E¹ in each instance is independently aryl-C₀₋₁₀alkyl,aryl-C₂₋₁₀alkenyl, aryl-C₂₋₁₀alkynyl, hetaryl-C₀₋₁₀alkyl,hetaryl-C₂₋₁₀alkenyl, or hetaryl-C₂₋₁₀alkynyl, where the attachmentpoint is from either the left or right as written, where any of which isoptionally substituted with one or more independent halo, —CF₃, —OCF₃,—OR³¹, —NR³¹R³², —C(O)R³¹, —CO₂R³¹, —C(═O)NR³¹R³², —NO₂, —CN,—S(O)₀₋₂R³¹, —S(O)₀₋₂NR³¹R³², —NR³¹C(═O)R³², —NR³¹C(═O)OR³²,—NR³¹C(═O)NR³²R³³, —NR³¹¹S(O)₀₋₂R³², —C(═S)OR³¹, —C(═O)SR³¹,—NR³¹C(═NR³²)NR³³R³¹, —NR³¹C(═NR³²)OR³³, —NR³¹C(═NR³²)SR³³, —OC(═O)OR³¹,—OC(═O)NR³¹, R³², —OC(═O)SR³¹, —SC(═O)OR³¹, or —SC(═O)NR³¹R³²substituents;

in the cases of —NR³¹R³², —NR³¹¹R³²¹, —NR³¹²R³²², —NR³³²R³⁴¹,—NR³¹³R³²³, and —NR³²³R³³³, the respective R³¹ and R³², R³¹¹ and R³²¹,R³¹² and R³²², R³³¹ and R³⁴¹, R³¹³ and R³²³, and R³²³ and R³³³ areoptionally taken together with the nitrogen atom to which they areattached to form a 3-10 membered saturated or unsaturated ring; whereinsaid ring in each instance independently is optionally substituted byone or more independent —N(C₀₋₈alkyl)(C₀₋₈alkyl), hydroxyl, halogen,oxo, aryl, hetaryl, C₀₋₆alkyl, —C₀₋₈alkylC₃₋₈cycloalkyl,—C₀₋₈alkyl-N(C₀₋₈alkyl)S(O)₀₋₂C₀₋₈alkyl,—C₀₋₈alkyl-N(C₀₋₈alkyl)S(O)₀₋₂N(C₀₋₈alkyl)(C₀₋₈alkyl),—C₀₋₈alkyl-N(C₀₋₈alkyl)CO₂(C₀₋₈alkyl),—C₀₋₈alkyl-CON((C₀₋₈alkyl))S(O)₀₋₂(C₀₋₈alkyl),—C₀₋₈alkyl-S(O)₀₋₂N(C₀₋₈alkyl)(C₀₋₈alkyl),—C₀₋₈alkyl-N(C₀₋₈alkyl)CO(C₀₋₈alkyl),—C₀₋₈alkyl-N(C₀₋₈alkyl)CON(C₀₋₈alkyl)(C₀₋₈alkyl),—C₀₋₈alkyl-CON(C₀₋₈alkyl)(C₀₋₈alkyl), —C₀₋₈alkyl-CO₂(C₀₋₈alkyl),—C₀₋₈alkylS(O)₀₋₂(C₀₋₈alkyl), —C₀₋₈alkyl-O—C₀₋₈alkyl,—C₀₋₈alkyl-O—C₀₋₈alkylcyclyl, —C₀₋₈alkyl-O—C₀₋₈alkylheterocycloalkyl,—C₀₋₈alkyl-O—C₀₋₈alkylaryl, —Oaryl, —C₀₋₈alkyl-O—C₀₋₈alkylhetaryl,—C₀₋₈alkyl-S—C₀₋₈alkyl, —C₀₋₈alkyl-S—C₀₋₈alkylC₃₋₈cycloalkyl,—C₀₋₈alkyl-S—C₀₋₈alkylheterocycloalkyl, —C₀₋₈alkyl-S—C₀₋₈alkylaryl,—C₀₋₈alkyl-S—C₀₋₈alkylhetaryl, —C₀₋₈alkyl-N(C₀₋₈alkyl)-C₀₋₈alkyl,—C₀₋₈alkyl-N(C₀₋₈alkyl)-C₀₋₈alkylC₃₋₈cycloalkyl,—C₀₋₈alkyl-N(C₀₋₈alkyl)-C₀₋₈alkylheterocycloalkyl,—C₀₋₈alkyl-N(C₀₋₈alkyl)-C₀₋₈alkylaryl,—C₀₋₈alkyl-N(C₀₋₈alkyl)-C₀₋₈alkylhetaryl,—C₀₋₈alkyl-N(C₀₋₈alkyl)(C₀₋₈alkyl), C₂₋₈alkenyl, C₂₋₈alkynyl, NO₂, CN,CF₃, OCF₃, or OCHF₂ substituents; wherein said ring in each instanceindependently optionally includes one or more heteroatoms other than thenitrogen;

m is 0, 1, 2, or 3;

n is 0, 1, 2, 3, or 4;

aa is 0 or 1; and

provided that Formula I is not

-   trans-4-[8-amino-1-(7-chloro-4-hydroxy-1H-indol-2-yl)imidazo[1,5-a]pyrazin-3-yl]cyclohexanecarboxylic    acid,-   cis-3-[8-amino-1-(7-chloro-1H-indol-2-yl)imidazo[1,5-a]pyrazin-3-yl]cyclobutanecarboxylic    acid,-   trans-4-{8-amino-1-[7-(3-isopropyl)phenyl-1H-indol-2-yl]imidazo[1,5-a]pyrazin-3-yl}cyclohexanecarboxylic    acid or-   trans-4-{8-amino-[7-(2,5-dichloro)phenyl-1H-indol-2-yl]imidazo[1,5-a]pyrazin-3-yl}cyclohexanecarboxylic    acid.

Specific examples of compounds encompassed by Formula I, that are mTORkinase inhibitors that inhibit mTOR by binding to and directlyinhibiting both mTORC1 and mTORC2 kinases, were prepared as described inthe following schemes and examples.

The following schemes, intermediates and examples serve to demonstratehow to synthesize compounds that can be used in the invention describedherein, but in no way limit the invention. Additionally, the followingabbreviations are used: Me for methyl, Et for ethyl, iPr or iPr forisopropyl, n-Bu for n-butyl, t-Bu for tert-butyl, Ac for acetyl, Ph forphenyl, 4Cl-Ph or (4Cl)Ph for 4-chlorophenyl, 4Me-Ph or (4Me)Ph for4-methylphenyl, (p-CH3O)Ph for p-methoxyphenyl, (p-NO2)Ph forp-nitrophenyl, 4Br-Ph or (4Br)Ph for 4-bromophenyl, 2-CF3-Ph or (2CF3)Phfor 2-trifluoromethylphenyl, DMAP for 4-(dimethylamino)pyridine, DCC for1,3-dicyclohexylcarbodiimide, EDC for1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, HOBt for1-hydroxybenzotriazole, HOAt for 1-hydroxy-7-azabenzotriazole, TMP fortetramethylpiperidine, n-BuLi for n-butyllithium, CDI for1,1′-carbonyldiimidazole, DEAD for diethyl azodicarboxylate, PS-PPh3 forpolystyrene triphenylphosphine, DIEA for diisopropylethylamine, DIAD fordiisopropyl azodicarboxylate, DBAD for di-tert-butyl azodicarboxylate,HPFC for high performance flash chromatography, rt or RT for roomtemperature, min for minute, h for hour, Bn for benzyl, and LAH forlithium aluminum hydride.

Accordingly, the following are compounds that are useful asintermediates in the formation of the mTOR inhibiting EXAMPLES.

The compounds of Formula I of this invention and the intermediates usedin the synthesis of the compounds of this invention were preparedaccording to the following methods. Method A was used when preparingcompounds of Formula I-AA

as shown below in Scheme 1:

Method A:

where Q¹ and R³ are as defined previously for compound of Formula I.

In a typical preparation of compounds of Formula I-AA, compound ofFormula II was reacted with ammonia in a suitable solvent. Suitablesolvents for use in the above process included, but were not limited to,ethers such as tetrahydrofuran (THF), glyme, and the like;dimethylformamide (DMF); dimethyl sulfoxide (DMSO); acetonitrile;alcohols such as methanol, ethanol, isopropanol, trifluoroethanol, andthe like; and chlorinated solvents such as methylene chloride (CH₂Cl₂)or chloroform (CHCl₃). If desired, mixtures of these solvents were used,however, the preferred solvents were isopropanol and a mixture of THFand isopropanol. The above process was carried out at temperaturesbetween about −78° C. and about 120° C. Preferably, the reaction wascarried out between 80° C. and about 120° C. The above process toproduce compounds of the present invention was preferably carried in asealed reaction vessel such as but not limited to a thick walled glassreaction vessel or a stainless steel Parr bomb. An excess amount of thereactant, ammonia, was preferably used.

The compounds of Formula II of Scheme 1 were prepared as shown below inScheme 2.

where Q¹ and R³ are as defined previously for compound of Formula I.

In a typical preparation of a compound of Formula II, an intermediate ofFormula III was treated with POCl₃ or the isolated “Vilsmeier salt”[CAS#33842-02-3] in a suitable solvent at a suitable reactiontemperature. Suitable solvents for use in the above process included,but were not limited to, ethers such as tetrahydrofuran (THF), glyme,and the like; acetonitrile; and chlorinated solvents such as methylenechloride (CH₂Cl₂) or chloroform (CHCl₃). If desired, mixtures of thesesolvents were used or no solvent was used. The preferred solventsincluded methylene chloride and acetonitrile. The above process wascarried out at temperatures between about −78° C. and about 120° C.Preferably, the reaction was carried out between 20° C. and about 95° C.The above process to produce compounds of the present invention waspreferably carried out at about atmospheric pressure although higher orlower pressures were used if desired. Substantially equimolar amounts ofreactants were preferably used although higher or lower amounts wereused if desired.

The compounds of Formula III of Scheme 2 were prepared as shown below inScheme 3:

where Q¹ and R³ are as defined previously for compound of Formula I andA¹=OH, alkoxy, or a leaving group such as a halogen or imidazole.

In a typical preparation, of a compound of Formula III, a compound ofFormula IV and compound of Formula V were reacted under suitable amidecoupling conditions. Suitable conditions include but are not limited totreating compounds of Formula IV and V (when A¹=OH) with couplingreagents such as DCC or EDC in conjunction with DMAP, HOBt, HOAt and thelike. Suitable solvents for use in the above process included, but werenot limited to, ethers such as tetrahydrofuran (THF), glyme, and thelike; dimethylformamide (DMF); dimethyl sulfoxide (DMSO); acetonitrile;halogenated solvents such as chloroform or methylene chloride. Ifdesired, mixtures of these solvents were used, however the preferredsolvents were methylene chloride and DMF. The above process was carriedout at temperatures between about 0° C. and about 80° C. Preferably, thereaction was carried out at about rt. The above process to producecompounds of the present invention was preferably carried out at aboutatmospheric pressure although higher or lower pressures were used ifdesired. Substantially equimolar amounts of reactants were preferablyused although higher or lower amounts were used if desired.Alternatively, compounds of Formula IV and V (where A¹=F, Cl, Br, I)were reacted with bases such as triethylamine or ethyldiisopropylamineand the like in conjunction with DMAP and the like. Suitable solventsfor use in this process included, but were not limited to, ethers suchas tetrahydrofuran (THF), glyme, and the like; dimethylformamide (DMF);dimethyl sulfoxide (DMSO); acetonitrile; halogenated solvents such aschloroform or methylene chloride. If desired, mixtures of these solventswere used, however the preferred solvent was methylene chloride. Theabove process was carried out at temperatures between about −20° C. andabout 40° C. Preferably, the reaction was carried out between 0° C. and25° C. The above process to produce compounds of the present inventionwas preferably carried out at about atmospheric pressure although higheror lower pressures were used if desired. Substantially equimolar amountsof compounds of Formula IV and V (where A¹=F, Cl, Br, I) and base andsubstoichiometric amounts of DMAP were preferably used although higheror lower amounts were used if desired. Additionally, other suitablereaction conditions for the conversion of a compound of Formula IV to acompound of Formula III can be found in Larock, R. C. ComprehensiveOrganic Transformations, 2nd ed.; Wiley and Sons: New York, 1999, pp1941-1949.

The compounds of Formula IV of Scheme 3 were prepared as shown below inScheme 4:

where Q¹ is as defined previously for compound of Formula I andA²=phthalimido or N₃.

In a typical preparation, of a compound of Formula IV, a compound ofFormula VI is reacted under suitable reaction conditions in a suitablesolvent. When A²=phthalimido, suitable conditions include treatment ofcompound of Formula VI with hydrazine in a suitable solvent. Suitablesolvents for use in the above process included, but were not limited to,ethers such as tetrahydrofuran (THF), glyme, and the like;dimethylformamide (DMF); dimethyl sulfoxide (DMSO); acetonitrile;halogenated solvents such as chloroform or methylene chloride; alcoholicsolvents such as methanol and ethanol. If desired, mixtures of thesesolvents may be used, however the preferred solvent was ethanol. Theabove process was carried out at temperatures between about 0° C. andabout 80° C. Preferably, the reaction was carried out at about 22° C.The above process to produce compounds of the present invention waspreferably carried out at about atmospheric pressure although higher orlower pressures were used if desired. Substantially equimolar amounts ofreactants were preferably used although higher or lower amounts wereused if desired. In the transformation of compound of Formula VI to IV,if A²=N₃, then one skilled in the art would recognize that typical azidereduction conditions could be employed, including but not limited toPPh₃ and water or hydrogenation in the presence of a metal catalyst suchas palladium.

The compounds of Formula VI of Scheme 4 were prepared as shown below inScheme 5:

where Q¹ is as defined previously for compound of Formula I andA²=phthalimido or N₃.

In a typical preparation of a compound of Formula VI (whenA²=phthalimido), a compound of Formula VII was reacted with aphthalimide under typical Mitsunobu conditions in a suitable solvent inthe presence of suitable reactants. Suitable solvents for use in theabove process included, but were not limited to, ethers such astetrahydrofuran (THF), glyme, and the like; dimethylformamide (DMF);dimethyl sulfoxide (DMSO); acetonitrile (CH₃CN); chlorinated solventssuch as methylene chloride (CH₂Cl₂) or chloroform (CHCl₃). If desired,mixtures of these solvents were used, however, the preferred solvent wasTHF. Suitable reactants for use in the above process included, but werenot limited to, triphenylphosphine and the like, and an azodicarboxylate(DIAD, DEAD, DBAD).

The preferred reactants were triphenylphosphine or resin-boundtriphenylphosphine (PS-PPh₃), and DIAD. The above process may be carriedout at temperatures between about −78° C. and about 100° C. Preferably,the reaction was carried out at about 22° C. The above process toproduce compounds of the present invention was preferably carried out atabout atmospheric pressure although higher or lower pressures were usedif desired. Substantially equimolar amounts of reactants were preferablyused although higher or lower amounts were used if desired. Generally,one equivalent or a slight excess, 1.1 equivalents, oftriphenylphosphine, DIAD and phthalimide was used per equivalent ofcompound of Formula VII. Additionally, compound of Formula VII can bereacted with Ts₂O, Ms₂O, Tf₂O, TsCl, MsCl, or SOCl₂ in which the hydroxygroup is converted to a leaving group such as its respective tosylate,mesylate, triflate, or halogen such as chloro and subsequently reactedwith an amine equivalent such as NH(Boc)₂, phthalimide, potassiumphthalimide, or sodium azide. Conversion of the amine equivalents byknown methods such as by treating under acidic conditions (NH(Boc)₂),with hydrazine (phthalimide) as shown in Scheme 4, or withtriphenylphosphine/water (azide) will afford the desired amine as shownin Scheme 4.

The compounds of Formula VII of Scheme 5 were prepared from aldehydesQ¹-CHO and a 2-chloropyrazine VIII as shown below in Scheme 6:

where Q¹ is as defined previously for compound of Formula I.

In a typical preparation, of a compound of Formula VII, a compound ofFormula VIII was reacted under suitable reaction conditions in asuitable solvent with a compound of Formula Q¹-CHO. Suitable conditionsincluded but were not limited to treating compounds of Formula VIII witha base such as lithium tetramethylpiperidide (Li-TMP) followed bytreating with compounds of Formula Q¹-CHO. Lithium tetramethylpiperididemay be prepared by reacting tetramethylpiperidine with n-butyllithium at−78° C. and warming up to 0° C. Suitable solvents for use in the aboveprocess included, but were not limited to, ethers such astetrahydrofuran (THF), glyme, and the like. Polar solvents such ashexamethylphosphoramide (HMPA),1,3-dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimidinone (DMPU), and the likemay be added if necessary. If desired, mixtures of these solvents wereused, however, the preferred solvent was THF. The above process may becarried out at temperatures between about −80° C. and about 20° C.Preferably, the reaction was carried out at −78° C. to 0° C. The aboveprocess to produce compounds of the present invention was preferablycarried out at about atmospheric pressure although higher or lowerpressures were used if desired. Substantially equimolar amounts ofreactants were preferably used although higher or lower amounts wereused if desired.

The compounds of Formula I of this invention and the intermediates usedin the synthesis of the compounds of this invention were also preparedaccording to the following methods. Method AA was used when preparingcompounds of Formula I-AA from compound of Formula I-AAA as shown belowin Scheme 7:

Method AA:

where Q¹ and R³ are as defined previously for compound of Formula I,A¹¹=halogen such as Cl, Br, or I and B(OR)₂=suitable boronic acid/ester.

In a typical preparation of compounds of Formula I-AA, compound ofFormula I-AAA was reacted with a suitable boronic acid/ester (Q¹-B(OR)₂)in a suitable solvent via typical Suzuki coupling procedures. Suitablesolvents for use in the above process included, but were not limited to,ethers such as tetrahydrofuran (THF), glyme, dioxane, dimethoxyethane,and the like; dimethylformamide (DMF); dimethyl sulfoxide (DMSO);acetonitrile; alcohols such as methanol, ethanol, isopropanol,trifluoroethanol, and the like; and chlorinated solvents such asmethylene chloride (CH₂Cl₂) or chloroform (CHCl₃). If desired, mixturesof these solvents were used, however, the preferred solvent wasdimethoxyethane/water. The above process was carried out at temperaturesbetween about −78° C. and about 120° C. Preferably, the reaction wascarried out between 60° C. and about 100° C. The above process toproduce compounds of the present invention was preferably carried out atabout atmospheric pressure although higher or lower pressures were usedif desired. Substantially equimolar amounts of reactants were preferablyused although higher or lower amounts were used if desired.

One skilled in the art will appreciate that alternative methods may beapplicable for preparing compounds of Formula I-AA from I-AAA. Forexample, compound of Formula I-AAA could be reacted with a suitableorganotin reagent Q¹-SnBu₃ or the like in a suitable solvent via typicalStille coupling procedures.

The compounds of Formula I-AAA of Scheme 7 were prepared as shown belowin Scheme 8.

where R³ is as defined previously for compound of Formula I andA¹¹=halogen such as Cl, Br, or I.

In a typical preparation of compounds of Formula I-AAA, compound ofFormula II-Z was reacted with ammonia in a suitable solvent. Suitablesolvents for use in the above process included, but were not limited to,ethers such as tetrahydrofuran (THF), glyme, and the like;dimethylformamide (DMF); dimethyl sulfoxide (DMSO); acetonitrile;alcohols such as methanol, ethanol, isopropanol, trifluoroethanol, andthe like; and chlorinated solvents such as methylene chloride (CH₂Cl₂)or chloroform (CHCl₃). If desired, mixtures of these solvents were used,however, the preferred solvents were isopropanol and a mixture of THFand isopropanol. The above process was carried out at temperaturesbetween about −78° C. and about 120° C. Preferably, the reaction wascarried out between 80° C. and about 120° C. The above process toproduce compounds of the present invention was preferably carried in asealed reaction vessel such as but not limited to a thick walled glassreaction vessel or a stainless steel Parr bomb. An excess amount of thereactant, ammonia, was preferably used.

The compounds of Formula II-Z of Scheme 8 were prepared as shown belowin Scheme 9.

where R³ is as defined previously for compound of Formula I andA¹¹=halogen such as Cl, Br, or I.

In a typical preparation of a compound of Formula II-Z, intermediateIII-Z was converted to compound of Formula II-Z′. Intermediate ofFormula III-Z was treated with POCl₃ in a suitable solvent at a suitablereaction temperature. Suitable solvents for use in the above processincluded, but were not limited to, ethers such as tetrahydrofuran (THF),glyme, and the like; acetonitrile; and chlorinated solvents such asmethylene chloride (CH₂Cl₂) or chloroform (CHCl₃). If desired, mixturesof these solvents were used. The preferred solvents included methylenechloride and acetonitrile. The above process was carried out attemperatures between about −78° C. and about 120° C. Preferably, thereaction was carried out between 20° C. and about 95° C. The aboveprocess to produce compounds of the present invention was preferablycarried out at about atmospheric pressure although higher or lowerpressures were used if desired. Substantially equimolar amounts ofreactants were preferably used although higher or lower amounts wereused if desired. In the conversion of compound of Formula III-Z toII-Z′, suitable halogenating agent were used, but were not limited to,Br₂, I₂, Cl₂, N-chlorosuccinimide, N-bromosuccinimide, orN-iodosuccinimide. The preferred halogenating agent wasN-iodosuccinimide. Suitable solvents for use in the above processincluded, but were not limited to, ethers such as tetrahydrofuran (THF),glyme, and the like; dimethylformamide (DMF); dimethyl sulfoxide (DMSO);acetonitrile; alcohols such as methanol, ethanol, isopropanol,trifluoroethanol, and the like; and chlorinated solvents such asmethylene chloride (CH₂Cl₂) or chloroform (CHCl₃). If desired, mixturesof these solvents were used, however, the preferred solvent was DMF. Theabove process was carried out at temperatures between about −78° C. andabout 120° C. Preferably, the reaction was carried out between 40° C.and about 75° C. The above process to produce compounds of the presentinvention was preferably carried out at about atmospheric pressurealthough higher or lower pressures were used if desired. Substantiallyequimolar amounts of reactants were preferably used although higher orlower amounts were used if desired.

The compounds of Formula III-Z of Scheme 9 were prepared as shown belowin Scheme 10:

where R³ is as defined previously for compound of Formula I and A¹=OH,alkoxy, or a leaving group such as chloro or imidazole.

In a typical preparation, of a compound of Formula III-Z, a compound ofFormula IV-Z and compound of Formula V were reacted under suitable amidecoupling conditions. Suitable conditions include but are not limited totreating compounds of Formula IV-Z and V (when A¹=OH) with couplingreagents such as DCC or EDC in conjunction with DMAP, HOBt, HOAt and thelike. Suitable solvents for use in the above process included, but werenot limited to, ethers such as tetrahydrofuran (THF), glyme, and thelike; dimethylformamide (DMF); dimethyl sulfoxide (DMSO); acetonitrile;halogenated solvents such as chloroform or methylene chloride. Ifdesired, mixtures of these solvents were used, however the preferredsolvent was methylene chloride. The above process was carried out attemperatures between about 0° C. and about 80° C. Preferably, thereaction was carried out at about 22° C. The above process to producecompounds of the present invention was preferably carried out at aboutatmospheric pressure although higher or lower pressures were used ifdesired. Substantially equimolar amounts of reactants were preferablyused although higher or lower amounts were used if desired.Additionally, if compound of Formula IV-Z was a salt or bis-salt, asuitable base was required and included, but was not limited to,diisopropylethylamine or triethylamine. Alternatively, compounds ofFormula IV-Z and V (where A¹=F, Cl, Br, I) were reacted with bases suchas triethylamine or ethyldiisopropylamine and the like in conjunctionwith DMAP and the like. Suitable solvents for use in this processincluded, but were not limited to, ethers such as tetrahydrofuran (THF),glyme, and the like; dimethylformamide (DMF); dimethyl sulfoxide (DMSO);acetonitrile; halogenated solvents such as chloroform or methylenechloride. If desired, mixtures of these solvents were used, however thepreferred solvent was methylene chloride. The above process was carriedout at temperatures between about −20° C. and about 40° C. Preferably,the reaction was carried out between 0° C. and 25° C. The above processto produce compounds of the present invention was preferably carried outat about atmospheric pressure although higher or lower pressures wereused if desired. Substantially equimolar amounts of compounds of FormulaIV-Z and V (where A¹=F, Cl, Br, I) and base and substoichiometricamounts of DMAP were preferably used although higher or lower amountswere used if desired. Additionally, other suitable reaction conditionsfor the conversion of an amine (compound of Formula IV-Z) to an amide(compound of Formula III-Z) can be found in Larock, R. C. ComprehensiveOrganic Transformations, 2nd ed.; Wiley and Sons: New York, 1999, pp1941-1949.

The compounds of Formula IV-Z of Scheme 10 were prepared as shown belowin Scheme 11:

where A² is phthalimido or N₃.

In a typical preparation, of a compound of Formula IV-Z, a compound ofFormula VI-Z is reacted under suitable reaction conditions in a suitablesolvent. When A²=phthalimido, suitable conditions include treatment ofcompound of Formula VI-Z with hydrazine in a suitable solvent. Suitablesolvents for use in the above process included, but were not limited to,ethers such as tetrahydrofuran (THF), glyme, and the like;dimethylformamide (DMF); dimethyl sulfoxide (DMSO); acetonitrile;halogenated solvents such as chloroform or methylene chloride; alcoholicsolvents such as methanol and ethanol. If desired, mixtures of thesesolvents may be used, however the preferred solvent was ethanol. Theabove process was carried out at temperatures between about 0° C. andabout 80° C. Preferably, the reaction was carried out at about 22° C.The above process to produce compounds of the present invention waspreferably carried out at about atmospheric pressure although higher orlower pressures were used if desired. Substantially equimolar amounts ofreactants were preferably used although higher or lower amounts wereused if desired.

The compounds of Formula VI-Z of Scheme 11 were prepared as shown belowin Scheme 12:

where A²=phthalimido or N₃.

In a typical preparation of a compound of Formula VI-Z (whenA²=phthalimido), a compound of Formula VII-Z was reacted with aphthalimide under typical Mitsunobu conditions in a suitable solvent inthe presence of suitable reactants. Suitable solvents for use in theabove process included, but were not limited to, ethers such astetrahydrofuran (THF), glyme, and the like; dimethylformamide (DMF);dimethyl sulfoxide (DMSO); acetonitrile (CH₃CN); chlorinated solventssuch as methylene chloride (CH₂Cl₂) or chloroform (CHCl₃). If desired,mixtures of these solvents were used, however, the preferred solvent wasTHF. Suitable reactants for use in the above process included, but werenot limited to, triphenylphosphine and the like, and an azodicarboxylate(DIAD, DEAD, DBAD). The preferred reactants were triphenylphosphine orresin-bound triphenylphosphine (PS-PPh₃) and DIAD. The above process maybe carried out at temperatures between about −78° C. and about 100° C.Preferably, the reaction was carried out at about 22° C. The aboveprocess to produce compounds of the present invention was preferablycarried out at about atmospheric pressure although higher or lowerpressures were used if desired. Substantially equimolar amounts ofreactants were preferably used although higher or lower amounts wereused if desired. Generally, 1.0 or 1.1 equivalents oftriphenylphosphine, DIAD and phthalimide was used per equivalent ofcompound of Formula VII-Z. Additionally, compound of Formula VII-Z canbe reacted with Ts₂O, Ms₂O, Tf₂O, TsCl, MsCl, or SOCl₂ in which thehydroxy group is converted to a leaving group such as its respectivetosylate, mesylate, triflate, or halogen such as chloro and subsequentlyreacted with an amine equivalent such as NH(Boc)₂, phthalimide,potassium phthalimide or sodium azide. Conversion of the amineequivalents by known methods such as by treating under acidic conditions(NH(Boc)₂), with hydrazine (phthalimide) as shown in Scheme 4, or withtriphenylphosphine/water (azide) will afford the desired amine as shownin Scheme 4.

The compounds of Formula VII-Z of Scheme 12 were prepared from2-chloropyrazine VIII as shown below in Scheme 13:

In a typical preparation, of a compound of Formula VII-Z, a compound ofFormula VIII was reacted under suitable reaction conditions in asuitable solvent. Suitable reaction conditions included, but were notlimited to, treating compounds of Formula VIII with a base such aslithium tetramethylpiperidide (Li-TMP) followed by treatment with areagent containing a carbonyl equivalent followed by treatment with asuitable reducing agent. Lithium tetramethylpiperidide may be preparedby reacting tetramethylpiperidine with n-butyllithium at −78° C. andwarming up to 0° C. Suitable solvents for use in the above processincluded, but were not limited to, ethers such as tetrahydrofuran (THF),glyme, and the like. Polar solvents such as hexamethylphosphoramide(HMPA), 1,3-dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimidinone (DMPU), andthe like may be added if necessary. If desired, mixtures of thesesolvents were used, however, the preferred solvent was THF. Suitablecarbonyl equivalent reagents include, but are not limited to, formamidessuch as DMF or suitable chloroformate such as methyl or ethylchloroformate. After addition of the suitable carbonyl equivalentreagent, the reaction if charged with a polar protic solvent such as,but not limited to, methanol or ethanol followed by treatment with asuitable reducing agent such as sodium borohydride. The above processmay be carried out at temperatures between about −80° C. and about 20°C. Preferably, the reaction was carried out at −78° C. to 0° C. Theabove process to produce compounds of the present invention waspreferably carried out at about atmospheric pressure although higher orlower pressures were used if desired. Substantially equimolar amounts ofreactants were preferably used although higher or lower amounts wereused if desired.

The compounds of Formula X-Z (Q¹-CHO) of Scheme 6 were prepared as shownbelow in Scheme 14:

where Q1 is as defined previously for compound of Formula I.

In a typical preparation, of a compound of Formula X-Z (Q¹-CHO), acompound of Formula IX-Z (Q¹-CH₃) was reacted with a suitable oxidizingagent under suitable reaction conditions. Suitable oxidizing agentsincluded, but were not limited to, selenium dioxide. Suitable reactionconditions for use in the above process included, but were not limitedto, heating a mixture of selenium dioxide and compounds of Formula IX-Z(Q¹-CH₃) neat or in a suitable solvent such as, but not limited to,chlorobenzene or sulpholane. The above process may be carried out attemperatures between about 120° C. and about 180° C. Preferably, thereaction was carried out at 150° C. to 165° C. The above process toproduce compounds of the present invention was preferably carried out atabout atmospheric pressure although higher or lower pressures were usedif desired. Preferably, 1-1.5 eq. selenium dioxide were used althoughhigher or lower amounts were used if desired. Alternatively, a compoundof Formula IX-Z (Q¹-CH₃) was reacted first with a halogenating agent anda radical initiator under suitable reaction conditions in a suitablesolvent to give a compound of Formula Q¹-CH₂-Hal (wherein Hal=Cl or Br)that was then further reacted with DMSO and a base under suitablereaction conditions to give a compound of Formula X-Z (Q¹-CHO). Suitablehalogenating agents included, but were not limited to, bromine,N-bromosuccinimide, and chlorine. Preferably, N-bromosuccinimide wasused. Suitable radical initiators included, but were not limited to,2,2′-azobisisobutyronitrile (AIBN) and UV light. Preferably, AIBN wasused. Preferably, carbon tetrachloride was used as solvent for thehalogenation step, although other halogenated solvents may be added. Thehalogenation may be carried out at temperatures between about 60° C. andabout 100° C. Preferably, the reaction was carried out at about 80° C.Suitable bases included, but were not limited to, sodiumhydrogencarbonate, sodium dihydrogenphosphate, disodiumhydrogenphosphate, and collidine. Preferably, sodium hydrogencarbonatewas used. DMSO was preferably used as solvent although other solventsmay be added. The second step may be carried out at temperatures betweenabout 40° C. and about 140° C. Preferably, the reaction was carried outat about 90° C. Additionally, other suitable reaction conditions for theconversion of Q¹-CH₃ to Q¹-CHO can be found in Larock, R. C.Comprehensive Organic Transformations, 2nd ed.; Wiley and Sons: NewYork, 1999, pp 1205-1207 and 1222-1224.

The compounds of Formula XIV-Z (Q¹-B(OR)₂) of Scheme 7 were prepared asshown below in Scheme 15:

where Q¹ is as defined previously for compound of Formula I, A¹¹¹=OTf orhalogen such as Cl, Br, or I and B(OR)₂=suitable boronic acid/ester.

In a typical preparation, of a compound of Formula XIV-Z (Q¹-B(OR)₂), acompound of Formula XIII-Z (Q¹-A¹¹¹) was reacted with a suitable metalcatalyst and a suitable boronating agent under suitable reactionconditions. Suitable metal catalyst agents included, but were notlimited to, Pd(OAc)₂ in the presence of1,3-bis(2,6-diisopropylphenyl)imidazolium chloride. Suitable boronatingagents included, but were not limited to, bis(pinacolato)diboron.Suitable reaction conditions for use in the above process included, butwere not limited to, heating a mixture of Pd(OAc)₂,1,3-bis(2,6-diisopropylphenyl)imidazolium chloride, KOAc, andbis(pinacol)borane in a suitable solvent such as, but not limited to,THF. The above process may be carried out at temperatures between about20° C. and about 100° C. Preferably, the reaction was carried out at 60°C. to 80° C. The above process to produce compounds of the presentinvention was preferably carried out at about atmospheric pressurealthough higher or lower pressures were used if desired. Preferably, 2-3eq. KOAc, 1-1.5 eq. bis(pinacol)borane, 0.03-1 eq. Pd(OAc)₂, and 0.09-3eq. 1,3-bis(2,6-diisopropylphenyl)imidazolium chloride were usedalthough higher or lower amounts were used if desired. Additionally,other suitable reaction conditions for the conversion of Q¹-A¹¹¹ toQ¹-B(OR)₂ can be found in the literature which involve a variety ofQ¹-A¹¹¹ or aryl/heteroarylhalides and a variety of conditions (Biooganic& Medicinal Chemistry Letters, 2003, 12(22), 4001; Biooganic & MedicinalChemistry Letters, 2003, 13(18), 3059; Chemical Communications(Cambridge, UK), 2003, 23, 2924; Synthesis, 2002, 17, 2503; AngewandteChemie, International Ed., 2002, 41(16), 3056; Journal of the AmericanChemical Society, 2002, 124(3), 390; Organic Letters, 2002, 4(4), 541;Tetrahedron, 2001, 57(49), 9813; Journal of Organic Chemistry, 2000,65(1), 164; Journal of Organic Chemistry, 1997, 62(19), 6458; Journal ofOrganometallic Chemistry, 1983, 259(3), 269). In some cases, compoundsof Formula XIII-Z (Q¹-A¹¹¹) and XIV-Z (Q¹-B(OR)₂) are commerciallyavailable or synthesized according to literature procedures. In caseswhere neither are available, compounds of Formula XIII-Z (Q¹-A¹¹¹) andXIV-Z (Q¹-B(OR)₂) were synthesized via procedures described in theexperimental section herein.

Both R³ and Q¹ in the compounds described herein in some instancescontain functional groups that can be further manipulated. It would beappreciated by those skilled in the art that such manipulation offunctional groups can be accomplished with key intermediates or withlate stage compounds. Such functional group transformations areexemplified in the following Schemes 16-26 as well as in theexperimental section but are in no way meant to limit the scope of suchtransformations. Additionally, the chemistry shown in Schemes 16-26 canalso be applied to compounds of I-AAA, II-Z, and II-Z′.

The compounds of Formula I-A (compounds of Formula I-AA whereR³═Z—CONR³¹²R³²²) were prepared as shown below in Scheme 17:

where Q¹, R³¹² and R³²² are as defined previously for compound ofFormula I and A³=hydrogen or alkyl such as methyl or ethyl.

In a typical preparation of compound of Formula I-A, when A³=alkyl andR³¹² and R³²² were both equal to H, reaction of compound of Formula II-A(compounds of Formula II where R³═Z—CO₂A³) with ammonia in a suitablesolvent, afforded compound of Formula I-A. Suitable solvents for use inthe above process included, but were not limited to, ethers such astetrahydrofuran (THF), glyme, and the like; dimethylformamide (DMF);dimethyl sulfoxide (DMSO); acetonitrile; alcohols such as methanol,ethanol, isopropanol, trifluoroethanol, and the like; and chlorinatedsolvents such as methylene chloride (CH₂Cl₂) or chloroform (CHCl₃). Ifdesired, mixtures of these solvents were used, however, the preferredsolvents were isopropanol and a mixture of isopropanol/THF. The aboveprocess was carried out at temperatures between about −78° C. and about120° C. Preferably, the reaction was carried out between 80° C. andabout 120° C. The above process to produce compounds of the presentinvention was preferably carried out at about atmospheric pressurealthough higher or lower pressures were used if desired. Substantiallyequimolar amounts of reactants were preferably used although higher orlower amounts were used if desired. Additionally, in a typicalpreparation of compound of Formula I-A, compound of Formula II-A (whenA³=H) was reacted with HNR³¹²R³²² followed by ammonia in a suitablesolvent. When A³=H, typical coupling procedures as described in Scheme 3(conversion of CO₂H to COCl via treatment with SOCl₂ or oxalyl chloridefollowed by reaction with HR³¹²R³²² or treatment of CO₂H and HR³¹²R³²²with EDC or DCC in conjunction with DMAP, HOBT, or HOAt and the like)were employed to afford the transformation of a carboxylic acid to anamide. When A³=alkyl such as methyl or ethyl, treatment of the esterwith Al(NR³¹²R³²²) afforded conversion of CO₂A³ to CO(NR³¹²R³²²).Subsequent treatment with ammonia afforded compounds of Formula I-A.

The compounds of Formula I-A′ (compounds of Formula I-AA whereR³═Z—CO₂A³) and I-A″ (compounds of Formula I-AA where R³═Z—CO₂H) wereprepared as shown below in Scheme 17:

where Q¹ is as defined previously for compounds of Formula I andA³=alkyl such as methyl or ethyl.

In a typical preparation of compound of Formula I-A′, compound ofFormula II-A was reacted with ammonia in a suitable solvent. Suitablesolvents for use in the above process included, but were not limited to,ethers such as tetrahydrofuran (THF), glyme, and the like;dimethylformamide (DMF); dimethyl sulfoxide (DMSO); acetonitrile;alcohols such as methanol, ethanol, isopropanol, trifluoroethanol, andthe like; and chlorinated solvents such as methylene chloride (CH₂Cl₂)or chloroform (CHCl₃). If desired, mixtures of these solvents were used,however, the preferred solvent was isopropanol. The above process wascarried out at temperatures between about −78° C. and about 120° C.Preferably, the reaction was carried out between 100° C. and about 120°C. The above process to produce compounds of the present invention waspreferably carried out at about atmospheric pressure although higher orlower pressures were used if desired. In most cases, the reactions wererun in a sealed tube. Substantially equimolar amounts of reactants werepreferably used although higher or lower amounts were used if desired.Typically, an excess of ammonia was used and the reaction was monitoredin order to ensure that additional of ammonia to the ester moiety didnot occur to an appreciable extent. Additionally, in a typicalpreparation of compound of Formula I-A″, compound of Formula I-A′ wasreacted under typical saponification conditions such as NaOH inTHF/H₂O/MeOH. Suitable solvents for use in the above process included,but were not limited to, ethers such as tetrahydrofuran (THF), glyme,and the like; dimethylformamide (DMF); dimethyl sulfoxide (DMSO);acetonitrile; alcohols such as methanol, ethanol, isopropanol,trifluoroethanol, and the like; and chlorinated solvents such asmethylene chloride (CH₂Cl₂) or chloroform (CHCl₃). If desired, mixturesof these solvents were used, however, the preferred solvent was amixture of THF/H₂O/MeOH. The above process was carried out attemperatures between about −78° C. and about 120° C. Preferably, thereaction was carried out between rt and about 60° C. The above processto produce compounds of the present invention was preferably carried outat about atmospheric pressure although higher or lower pressures wereused if desired. Substantially equimolar amounts of reactants werepreferably used although higher or lower amounts were used if desired.

The compounds of Formula II-B (compounds of Formula II where R³═Z—CH₂OH)and I-B (compounds of Formula I-AA where R³═Z—CH₂OH) were prepared asshown below in Scheme 18:

where Q¹ is as defined previously for compound of Formula I andA³=hydrogen or alkyl such as methyl or ethyl.

In a typical preparation of compound of Formula I-B, compound of FormulaII-A is treated with a suitable reducing agent such as lithium aluminumhydride in a suitable solvent, such as THF to afford compound of FormulaII-B. Suitable solvents for use in the above process included, but werenot limited to, ethers such as tetrahydrofuran (THF), glyme, and thelike; dimethylformamide (DMF); dimethyl sulfoxide (DMSO); acetonitrile;alcohols such as methanol, ethanol, isopropanol, trifluoroethanol, andthe like; and chlorinated solvents such as methylene chloride (CH₂Cl₂)or chloroform (CHCl₃). If desired, mixtures of these solvents were used.The preferred solvent was THF. The above process was carried out attemperatures between about −78° C. and about 120° C. Preferably, thereaction was carried out between 0° C. and about 50° C. The aboveprocess to produce compounds of the present invention was preferablycarried out at about atmospheric pressure although higher or lowerpressures were used if desired. Substantially equimolar amounts ofreactants were preferably used although higher or lower amounts wereused if desired. Subsequent treatment of compound of Formula II-B underpreviously described ammonolysis conditions (ammonia in isopropanol in asealed tube at 120° C.), afforded compound of Formula I-B.

The compounds of Formula II-C (compounds of Formula II whereR³═Z—CH₂A⁴), II-D (compounds of Formula II whereR³═Z—CH₂A⁵(R³¹³)(R³²³)_(aa)), I-B (compounds of Formula I-AA whereR³═Z—CH₂OH) and I-C (compounds of Formula I-AA whereR³═Z—CH₂A⁵(R³¹³)(R³²³)_(aa)) were prepared as shown below in Scheme 19:

where Q¹, R³¹³, and R³²³ are as defined previously for compound ofFormula I; A⁴=suitable leaving group such as OTs, OMs, OTf, or halo suchas chloro, bromo, or iodo; d=0 or 1; and A⁵=N, O or S.

In a typical preparation of compound of Formula I-C, the hydroxy groupof compound of Formula II-B was converted to a suitable leaving group,A⁴, such as Cl or OTs, OMs, or OTf, by reaction with SOCl₂ or Ts₂O,Ms₂O, or Tf₂O to afford compound of Formula II-C. Reaction of compoundof Formula II-C with HA⁵(R³¹³)(R³²³)_(aa) afforded compound of FormulaII-D. Subsequent reaction of compound of Formula II-D under previouslydescribed ammonolysis conditions afforded compound of Formula I-C.Additionally, compound of Formula II-B was converted to compound ofFormula I-B as described previously in Scheme 18. Further conversion ofcompound of Formula I-B to compound of Formula I-C was accomplished byfollowing the previously described conditions for the conversion ofcompound of Formula II-B to compound of Formula II-C and the furtherconversion of compound of Formula II-C to compound of Formula II-D (inthe net conversion of OH to A⁵(R³¹³)(R³²³)_(aa)). Furthermore, compoundof Formula II-B can be directly converted to compound of Formula II-D bytreating compound of Formula II-B with various alkylating agent or withphenols via the Mitsunobu reaction to afford compounds Formula II-D(compounds of Formula II where R³═CH₂—Z-A⁵(R³¹³)(R³²³)_(aa)) in whichA⁵=O, aa=0, and R³¹³=alkyl or aryl).

The compounds of Formula I-C′ (compounds of Formula I-AA whereR³═Z—CH₂-A²), I-C″ (compounds of Formula I-AA where R³═Z—CH₂—NH₂), andI-C′″ (compounds of Formula I-AA where R³═Z—CH₂—N(R³¹³)(R³²³)) wereprepared as shown below in Scheme 20:

where Q¹, R³¹³, and R³²³ are as defined previously for compound ofFormula I and A²=phthalimido or N₃.

In a typical preparation of compounds of Formula I-C′, I-C″, and I-C′″,the hydroxy group of compound of Formula I-B was converted to A²,following the procedures as described in Scheme 5 for the conversion ofcompound of Formula VII to compound of Formula VI. Reaction of compoundof Formula I-C′ under conditions described in Scheme 4 afforded compoundof Formula I-C″. Reaction of compound of Formula I-C″ with, but notlimited to various alkylating agents, various aldehydes/ketones underreductive amination conditions, various acylating agents such as aceticanhydride, benzoyl chlorides, or with carboxylic acids in the presenceof EDC or DCC with HOBT or HOAT, or with sulphonylating agents such asTs₂O or MeSO₂Cl afforded compounds of Formula I-C′″. For example, in atypical preparation of compounds of Formula I-C′″, a compound of FormulaI-C″ is treated with a suitable acylating agent in the presence of asuitable base in a suitable solvent. Suitable solvents for use in theabove process included, but were not limited to, ethers such astetrahydrofuran (THF), glyme, and the like; and chlorinated solventssuch as methylene chloride (CH₂Cl₂) or chloroform (CHCl₃). If desired,mixtures of these solvents were used, however, the preferred solvent waschloroform. Suitable bases for use in the above process included, butwere not limited to, trialkylamines such as diisopropylethylamine,triethylamine, or resin bound trialkylamines such as PS-DIEA. Thepreferred base was PS-DIEA. In the case where the suitable acylatingagent was acetic anhydride, the conversion of compound of Formula I-C″to compound of Formula I-C′″ where R³¹³═H and R³²³═COCH₃ wasaccomplished. The above process was carried out at temperatures betweenabout −78° C. and about 120° C. Preferably, the reaction was carried outbetween 0° C. and about 20° C. The above process to produce compounds ofthe present invention was preferably carried out at about atmosphericpressure although higher or lower pressures were used if desired.Substantially equimolar amounts of reactants were preferably usedalthough higher or lower amounts were used if desired.

The compounds of Formula I-D (compounds of Formula I-AA whereR³═(CH₂)_(n)—Z²—H and Z² is a heterocyclyl ring containing a nitrogenatom connected to H) and I-E (compounds of Formula I-AA whereR³═(CH₂)_(n)—Z²—R³¹ and Z² is a heterocyclyl ring containing a nitrogenatom connected to R³¹¹) were prepared as shown below in Scheme 21:

where Q¹ and R³¹ are as defined previously for compound of Formula I,G^(99a) is C(═O)A⁶ or CO₂A⁶, n=0-5, and A⁶=alkyl, aryl, or aralkyl.

In a typical preparation of compound of Formula I-E, compound of FormulaII-E is treated with suitable reagents capable of converting N-G^(99a)to N—H and therefore afford compound of Formula I-D. For example,treatment of compound of Formula II-E (when G^(99a) is equal to CO₂Bn)under previously described ammonolysis conditions followed by treatmentwith concentrated HCl and a suitable basic workup, affords compound ofFormula I-D. Compound of Formula I-D can be subjected to variousconditions including but not limited to reductive aminations,alkylations and ar(hetar)ylations, and acylations to afford amides,ureas, guanidines, carbamates, thiocarbamates, sulphonamides, andvariously substituted nitrogen adducts to afford the net conversion ofNH to NR².

The compounds of Formula II-G (compounds of Formula II where R³═Z³—OH),II-H (compounds of Formula II where R³═Z-A⁵(R³¹³)(R³²³)_(aa)), I-F(compounds of Formula I-AA where R³═Z—OH), and I-G (compounds of FormulaI-AA where R³═Z A⁵(R³¹³)(R³)_(aa)) were prepared as shown below inScheme 22:

where Q¹, R³¹³, and R³²³ are as defined previously for compound ofFormula I; aa=0 or 1; and A⁵=N, O or S.

In a typical preparation of compound of Formula I-F and I-G, thefollowing transformations occurred: Compound of Formula II-F was reducedwith a suitable reducing agent in a suitable solvent, such as sodiumborohydride in methanol to afford compound of Formula II-G. Compound ofFormula II-G was subjected to previously described ammonolysisconditions to afford compound of Formula I-F. Additionally, compounds ofFormula II-F can be reacted with various amines under reductiveamination conditions (NaBH₃CN or NaBH(OAc)₃ with HA⁵(R³¹³)(R³²³)_(aa)where d=0, A⁵=N, and R³¹³ and R³²³ are as previously described forcompound of Formula I) to afford compounds of Formula II-H where d=0,A⁵=N, and R³¹³ and R³²³ are as previously described for compound ofFormula I. Subsequent reaction of compounds of Formula II-H (compoundsof Formula II where R³═Z-A⁵(R³¹³)(R³²³)_(aa) where d=0, A⁵=N, and R³¹³and R³²³ are as previously described for compound of Formula I) withpreviously described ammonolysis conditions afforded compounds ofFormula I-G. Furthermore, compounds of Formula II-H from II-G and I-Gfrom I-F can be synthesized according to the conditions described inScheme 19 for the transformations of II-B to II-D and I-B to I-C,respectively.

The compounds of Formula I-C′″ (compounds of Formula I-AA whereR³═Z—CH₂—N(R³¹³)(R³²³)) were prepared as shown below in Scheme 23:

where Q¹, R³¹³, and R³²³ are as defined previously for compound ofFormula I and A⁴=suitable leaving group such as Cl, OTs, OMs or OTf.

In a typical preparation of compound of Formula I-C′″ (compounds ofFormula I-AA where R³═Z—CH₂—N(R³¹³)(R³²³)), the followingtransformations occurred: Compounds of Formula II-J (compounds ofFormula II where R³═Z═CH₂) were reacted with a suitable hydroboratingagent such as diborane, 9-borabicyclo[3.3.1]nonane (9-BBN),catecholborane and the like, in a suitable solvent such as THF followedby treatment with an suitable oxidizing agent such as hydrogen peroxidein basic aqueous solution or NaBO₃.H₂O to afford compounds of FormulaII-B. Further reaction of compounds of Formula II-B with previouslydescribed ammonolysis conditions afforded compounds of Formula I-B. Thehydroxy group of compounds of Formula I-B was then converted to asuitable leaving group, A⁴, such OTs, OMs, or OTf, by reaction withTs₂O, Ms₂O, or Tf₂O, respectively, to afford compounds of Formula I-H.Further reaction of compounds of Formula I-H with HN(R³¹³)(R³²³) whereR³¹³ and R³²³ are as previously described for compounds of Formula Iafforded compound of Formula I-C′″ (compounds of Formula I-AA whereR³═Z—CH₂—N(R³¹³)(R³²³)).

The compounds of Formula I-J (compounds of Formula I-AA whereR³═Z—OH(CH₂OH)), I-K (compounds of Formula I-AA where R³═Z═O), and I-L(compounds of Formula I-AA where R³═Z—NR³¹³R³²³) were prepared as shownbelow in Scheme 24:

where Q¹, R³¹² and R³²² are as defined previously for compound ofFormula I.

In a typical preparation of compound of Formula I-J (compounds ofFormula I-AA where R³═Z—OH(CH₂OH)), I-K (compounds of Formula I-AA whereR³═Z═O), and I-L (compounds of Formula I-AA where R³═Z—NR³¹²R³²²)compound of Formula II-J was treated under (compounds of Formula IIwhere R³═Z═CH₂) was reacted with a suitable dihydroxylating agent suchas osmium tetraoxide in the presence of NMO in a suitable solvent suchas THF to afford compound of Formula II-K (compounds of Formula II whereR³═Z—OH(CH₂OH)) as a mixture of cis and trans isomers. Compounds ofFormula II-K (compounds of Formula II where R³═Z—OH(CH₂OH)) were treatedwith a suitable oxidizing agent, such as but not limited to, NaIO₄,converting the diol into a ketone moiety, affording compound of FormulaII-L (compounds of Formula II where R³═Z═O). Compound of Formula II-L(compounds of Formula II where R³═Z═O) was then treated under typicalreductive amination conditions, involving a suitable amine, HNR³¹²R³²²and a suitable reducing agent, such as but not limited to, NaBH(OAc)₃ orNaBH(CN)₃, affording compound of Formula II-M (compounds of Formula IIwhere R³═Z—NR³¹²R³²²). Compound of Formula II-M (compounds of Formula IIwhere R³═Z—NR³¹²R³²²) was treated under ammonolysis conditions, ammoniain isopropanol in a stainless steel bomb at 110° C., to afford compoundof Formula I-L (compounds of Formula I-AA where R³═Z—NR³¹²R³²²).Moreover, compound of Formula II-K (compounds of Formula II whereR³═Z—OH(CH₂OH)) was treated under the ammonolysis conditions describedabove to afford compound of Formula I-J (compounds of Formula I-AA whereR³═Z—OH(CH₂OH)) as a mixture of isomers. Compound of Formula I-J(compounds of Formula I-AA where R³═Z—OH(CH₂OH)) was treated with asuitable oxidizing agent, such as but not limited to, NaIO₄, convertingthe diol into a ketone moiety, affording compound of Formula I-K(compounds of Formula I-AA where R³═Z═O), which was treated under thetypical reductive amination conditions described above to affordcompound of Formula I-L (compounds of Formula I-AA whereR³═Z—NR³¹²R³²²).

The compounds of Formula I-N (compounds of Formula I-AA whereR³═Z—OH(CH₂NR³¹³R³²³)) were prepared as shown below in Scheme 25:

where Q¹, R³¹³, and R³²³ are as defined previously for compound ofFormula I; A⁴=suitable leaving group such as OTs, OMs, or OTf.

In a typical preparation of compounds of Formula I-N (compounds ofFormula I-AA where R³═Z—OH(CH₂NR³¹³R³²³)), the primary hydroxyl group ofcompound of Formula I-J (compounds of Formula I-AA where R³═Z═OH(CH₂OH))was converted to a suitable leaving group, A⁴, such as OTs, OMs, or OTf,by reaction with Ts₂O, Ms₂O, or Tf₂O in the presence of a suitable basesuch as diisopropylamine or pyridine and solvent such as THF ormethylene chloride to afford compound of Formula I-M (compounds ofFormula I-AA where R³═Z═OH(CH₂A⁴)). Reaction of compound of Formula I-M(compounds of Formula I-AA where R³═Z—OH(CH₂A⁴)) with HN(R³¹³)(R³²³) ina suitable solvent such as THF or methylene chloride afforded compoundof Formula I-N (compounds of Formula I where R³═Z—OH(CH₂NR³¹³R³²³)).

The compounds of Formula I-O (compounds of Formula I whereR³═Z³—OH(G¹¹)) were prepared as shown below in Scheme 26:

where Q¹ and G¹¹ are as defined previously for compound of Formula I.

In a typical preparation of compounds of Formula I-O (compounds ofFormula I where R³═Z—OH(G¹¹)), the ketone moiety of compound of FormulaII-L (compounds of Formula II where R³═Z═O) was reacted with a suitablenucleophilic reagent such as MeMgBr or MeLi in a suitable solvent suchas THF to afford compound of Formula II-N (compounds of Formula II whereR³═Z—OH(G¹¹)). Compound of Formula II-N (compounds of Formula II whereR³═Z—OH(G¹)) was reacted under ammonolysis conditions, ammonia inisopropanol in a stainless steel bomb at 110° C., to afford compound ofFormula I-O (compounds of Formula I where R³═Z—OH(G¹¹)). Additionally,compound of Formula I-O (compounds of Formula I where R³═Z—OH(G¹¹)) wasprepared by reacting compound of Formula I-K (compounds of Formula I-AAwhere R³═Z═O) with a suitable nucleophilic reagent such as MeMgBr orMeLi in a suitable solvent such as THF.

The conversion of compounds of Formula I-PP′ and I-P′ to compounds ofFormula I-RR an I-R, respectively may be accomplished by reaction with aboronic acid ester using so-called “Liebeskind-Srogl” conditions such asthose described in Organic Letters, (2002), 4(6), 979 or Synlett,(2002), (3), 447.

A compound of Formula I-AB is equal to compound of Formula I whereinX₁═CH, X₂, X₄ and X₅═N, and X₃, X₆ and X₇═C; Q¹ is as defined for acompound of Formula I; R³ is C₀₋₁₀alkyl, cycloC₃₋₁₀alkyl,aminomethylcycloC₃₋₁₀alkyl, bicycloC₅₋₁₀alkyl, aryl, heteroaryl,aralkyl, heteroaralkyl, heterocyclyl, heterobicycloC₅₋₁₀alkyl,spiroalkyl, or heterospiroalkyl, any of which is optionally substitutedby one or more independent G¹¹ substituents; and G¹¹ is as defined for acompound of Formula I:

Method AB was used when preparing compounds of Formula I-AB as shownbelow in Scheme 28:

Method AB:

where Q¹ and R³ are as defined previously for compound of Formula I-AB,A¹=halogen such as Cl, Br, or I, and Q¹-B(OR)₂=suitable boronicacid/ester.

In a typical preparation of compounds of Formula I-AB, compound ofFormula I-ABA was reacted with a suitable boronic acid/ester of FormulaXIV-Z (Q¹-B(OR)₂) in a suitable solvent via typical Suzuki couplingprocedures. Suitable solvents for use in the above process included, butwere not limited to, ethers such as tetrahydrofuran (THF), glyme, andthe like; dimethylformamide (DMF); dimethyl sulfoxide (DMSO);acetonitrile; alcohols such as methanol, ethanol, isopropanol,trifluoroethanol, and the like; and chlorinated solvents such asmethylene chloride (CH₂Cl₂) or chloroform (CHCl₃). If desired, mixturesof these solvents were used, however, the preferred solvent systems wereTHF/water and DMF/water. The above process was carried out attemperatures between about 20° C. and about 120° C. Preferably, thereaction was carried out between 80° C. and about 100° C. The aboveprocess to produce compounds of the present invention was preferablycarried out at about atmospheric pressure although higher or lowerpressures were used if desired. Substantially equimolar amounts ofreactants were preferably used although higher or lower amounts wereused if desired.

One skilled in the art will appreciate that alternative methods may beapplicable for preparing compounds of Formula I-AB from I-ABA. Forexample, compound of Formula I-ABA could be reacted with a suitableorganotin reagent Q¹-SnBu₃ or the like in a suitable solvent via typicalStille coupling procedures.

The compounds of Formula I-ABA wherein R³ is C₁₋₁₀alkyl,cycloC₃₋₁₀alkyl, bicycloC₅₋₁₀alkyl, aralkyl, heteroaralkyl,heterocyclyl, heterobicycloC₅₋₁₀alkyl, spiroalkyl, or heterospiroalkyl,any of which is optionally substituted by one or more independent G¹¹substituents, of Scheme 28 were prepared as shown below in Scheme 29:

where R³ is C₁₋₁₀alkyl, cycloC₃₋₁₀alkyl, bicycloC₅₋₁₀alkyl, aralkyl,heteroaralkyl, heterocyclyl, heterobicycloC₅₋₁₀alkyl, spiroalkyl, orheterospiroalkyl, any of which is optionally substituted by one or moreindependent G¹¹ substituents; G¹¹ is as defined previously for compoundof Formula I, and A¹¹=halogen such as Cl, Br, or I.

In a typical preparation of a compound of Formula I-ABA, a compound ofFormula I-ABB was reacted with an alcohol R³—OH under typical Mitsunobuconditions in a suitable solvent in the presence of suitable reactants.Suitable solvents for use in the above process included, but were notlimited to, ethers such as tetrahydrofuran (THF), glyme, and the like;dimethylformamide (DMF); dimethyl sulfoxide (DMSO); acetonitrile(CH₃CN); chlorinated solvents such as methylene chloride (CH₂Cl₂) orchloroform (CHCl₃). If desired, mixtures of these solvents were used,however, the preferred solvent was THF. Suitable reactants for use inthe above process included, but were not limited to, triphenylphosphineand the like, and an azodicarboxylate (DIAD, DEAD, DBAD). The preferredreactants were triphenylphosphine or resin-bound triphenylphosphine andDIAD. The above process may be carried out at temperatures between about−78° C. and about 100° C. Preferably, the reaction was carried outbetween about 0° C. and 25° C. The above process to produce compounds ofthe present invention was preferably carried out at about atmosphericpressure although higher or lower pressures were used if desired.Substantially equimolar amounts of reactants were preferably usedalthough higher or lower amounts were used if desired. Generally, oneequivalent of triphenylphosphine, DIAD, and R³—OH was used perequivalent of compound of Formula I-ABB.

Alternatively, the compounds of Formula I-ABA may be prepared byalkylating compounds of Formula I-ABB with an alkylating agent R³-LG,wherein LG is a leaving group including, but not limited to, chloride,bromide, iodide, tosylate, mesylate, trifluoromethanesulfonate, undertypical alkylation conditions known to someone skilled in the art.

Preferably, in compounds of Formula I-ABB, A¹¹=Br and I. These compoundsare known (A¹¹=I: H. B. Cottam et al., J. Med. Chem. 1993, 36(22),3424-3430; A¹¹=Br: T. S. Leonova et al., Khim. Geterotsikl. Soedin.1982, (7), 982-984).

Compound of Formula I-AC is equal to compound of Formula I wherein X₁and X₅═CH, X₂ and X₄═N, and X₃, X₆ and X₇═C; Q¹ is as defined for acompound of Formula I; R³ is C₀₋₁₀alkyl, cycloC₃₋₁₀alkyl,bicycloC₅₋₁₀alkyl, aryl, heteroaryl, aralkyl, heteroaralkyl,heterocyclyl, heterobicycloC₅₋₁₀alkyl, spiroalkyl, or heterospiroalkyl,any of which is optionally substituted by one or more independent G¹¹substituents; and G¹¹ is as defined for a compound of Formula I:

Method AC was used when preparing compounds of Formula I-AB as shownbelow in Scheme 30:

Method AC:

where Q¹ and R³ are as defined previously for compound of Formula I-AC,A¹¹=halogen such as Cl, Br, or I and Q¹-B(OR)₂=suitable boronicacid/ester.

In a typical preparation of compounds of Formula I-AC, compound ofFormula I-ACA was reacted with a suitable boronic acid/ester XIV-Z(Q¹-B(OR)₂) in a suitable solvent via typical Suzuki couplingprocedures. Suitable solvents for use in the above process included, butwere not limited to, ethers such as tetrahydrofuran (THF), glyme, andthe like; dimethylformamide (DMF); dimethyl sulfoxide (DMSO);acetonitrile; alcohols such as methanol, ethanol, isopropanol,trifluoroethanol, and the like; and chlorinated solvents such asmethylene chloride (CH₂Cl₂) or chloroform (CHCl₃). If desired, mixturesof these solvents were used, however, the preferred solvent systems wereTHF/water and DMF/water. The above process was carried out attemperatures between about 20° C. and about 120° C. Preferably, thereaction was carried out between 80° C. and about 100° C. The aboveprocess to produce compounds of the present invention was preferablycarried out at about atmospheric pressure although higher or lowerpressures were used if desired. Substantially equimolar amounts ofreactants were preferably used although higher or lower amounts wereused if desired.

One skilled in the art will appreciate that alternative methods may beapplicable for preparing compounds of formula I-AC from I-ACA. Forexample, compound of Formula I-ACA could be reacted with a suitableorganotin reagent Q¹-SnBu₃ or the like in a suitable solvent via typicalStille coupling procedures.

The compounds of Formula I-ACA of Scheme 30 were prepared as shown belowin Scheme 31:

where R³ is as defined previously for compound of Formula I-AC, andA¹¹=halogen such as Cl, Br, or I.

In a typical preparation of compounds of Formula I-ACA, compound ofFormula XV was reacted with ammonia in a suitable solvent. Suitablesolvents for use in the above process included, but were not limited to,ethers such as tetrahydrofuran (THF), glyme, and the like;dimethylformamide (DMF); dimethyl sulfoxide (DMSO); acetonitrile;alcohols such as methanol, ethanol, isopropanol, trifluoroethanol, andthe like; and chlorinated solvents such as methylene chloride (CH₂Cl₂)or chloroform (CHCl₃). If desired, mixtures of these solvents were used,however, the preferred solvent was isopropanol. The above process wascarried out at temperatures between about −78° C. and about 120° C.Preferably, the reaction was carried out between 80° C. and about 100°C. The above process to produce compounds of the present invention waspreferably carried out in a glass pressure tube or a stainless steelreactor. Preferably, an excess of ammonia was used.

The compounds of Formula XVA (=compounds of Formula XV of Scheme 31wherein R³ is C₁₋₁₀alkyl, cycloC₃₋₁₀alkyl, bicycloC₅₋₁₀alkyl, aralkyl,heteroaralkyl, heterocyclyl, heterobicycloC₅₋₁₀alkyl, spiroalkyl, orheterospiroalkyl, any of which is optionally substituted by one or moreindependent G¹¹ substituents) were prepared as shown below in Scheme 32:

where R³ is C₁₋₁₀alkyl, cycloC₃₋₁₀alkyl, bicycloC₅₋₁₀alkyl, aralkyl,heteroaralkyl, heterocyclyl, heterobicycloC₅₋₁₀alkyl, spiroalkyl, orheterospiroalkyl, any of which is optionally substituted by one or moreindependent G¹¹ substituents; G¹¹ is as defined previously for compoundof Formula I; and A¹¹=halogen such as Cl, Br, or I.

In a typical preparation of a compound of Formula XVA, a compound ofFormula XVI was reacted with an alcohol R³—OH under typical Mitsunobuconditions in a suitable solvent in the presence of suitable reactants.Suitable solvents for use in the above process included, but were notlimited to, ethers such as tetrahydrofuran (THF), glyme, and the like;dimethylformamide (DMF); dimethyl sulfoxide (DMSO); acetonitrile(CH₃CN); chlorinated solvents such as methylene chloride (CH₂Cl₂) orchloroform (CHCl₃). If desired, mixtures of these solvents were used,however, the preferred solvent was THF. Suitable reactants for use inthe above process included, but were not limited to, triphenylphosphineand the like, and an azodicarboxylate (DIAD, DEAD, DBAD). The preferredreactants were triphenylphosphine or resin-bound triphenylphosphine andDIAD. The above process may be carried out at temperatures between about−78° C. and about 100° C. Preferably, the reaction was carried outbetween about 0° C. and 25° C. The above process to produce compounds ofthe present invention was preferably carried out at about atmosphericpressure although higher or lower pressures were used if desired.Substantially equimolar amounts of reactants were preferably usedalthough higher or lower amounts were used if desired. Generally, oneequivalent of triphenylphosphine, DIAD, and R³—OH was used perequivalent of compound of Formula XVI.

Alternatively, the compounds of Formula XVA may be prepared byalkylating compounds of Formula XVI with an alkylating agent R³-LG,wherein LG is a leaving group including, but not limited to, chloride,bromide, iodide, tosylate, mesylate, trifluoromethanesulfonate, undertypical alkylation conditions known to someone skilled in the art.

The compounds of Formula XVB (=compounds of Formula XV of Scheme 31wherein R³ is aryl or heteroaryl, optionally substituted by one or moreindependent G¹¹ substituents) were prepared as shown below in Scheme 33:

where R³ is aryl or heteroaryl, optionally substituted by one or moreindependent G¹¹ substituents, G¹¹ is as defined previously for compoundof Formula I; and A¹¹=halogen such as Cl, Br, or I.

In a typical preparation of compounds of Formula XVB, compound ofFormula XVI was reacted with a suitable boronic acid of FormulaR³—B(OH)₂ in a suitable solvent via typical copper(II)-mediated couplingprocedures. Suitable solvents for use in the above process included, butwere not limited to, ethers such as tetrahydrofuran (THF), glyme,1,4-dioxane, and the like; dimethylformamide (DMF);N-methylpyrrolidinone (NMP); chlorinated solvents such as methylenechloride (CH₂Cl₂). If desired, mixtures of these solvents were used,however, the preferred solvent was methylene chloride (CH₂Cl₂). Suitablereactants for use in the above process included, but were not limitedto, copper(II) acetate (Cu(OAc)₂), copper(II) triflate (Cu(OTf)₂), andthe like, and a base (pyridine, and the like). The preferred reactantswere Cu(OAc)₂ and pyridine. The above process to produce compounds ofthe present invention was preferably carried out at about atmosphericpressure under air, although higher or lower pressures could be used ifdesired. Preferably, the reaction was carried out at about 22° C.Generally, 1.5 eq. of copper(II) acetate, 2 eq. of pyridine, and 2 eq.of boronic acid of Formula R³—B(OH)₂ were used per equivalent ofcompound of Formula XVI.

All compounds of Formula XVI are known in the literature (A¹¹=I: L. B.Townsend et al., J. Med. Chem. 1990, 33, 1984-92; A¹¹=Br, Cl: L. B.Townsend et al., J. Med. Chem. 1988, 31, 2086-2092). Preferably, A¹¹=Brand I.

Both R³ and Q¹ in the compounds described herein in some instancescontain functional groups that can be further manipulated. It would beappreciated by those skilled in the art that such manipulation offunctional groups could be accomplished with key intermediates or withlate stage compounds. Such functional group transformations areexemplified in the following Schemes 34-35 as well as in theexperimental section but are in no way meant to limit the scope of suchtransformations.

The compounds of Formula I-ACA′ (=compounds of Formula I-ACA whereR³═Z—CONR³¹²R³²²) were prepared from compounds of Formula XV′(=compounds of Formula XV where R³═Z—CO₂A³) as shown below in Scheme 34:

where R³¹² and R³²² are as defined previously for compound of Formula I;A¹¹=halogen such as Cl, Br, or I; and A³=hydrogen or alkyl such asmethyl or ethyl.

In a typical preparation of compound of Formula I-ACA′, when A³=alkyland R³¹² and R³²² were both equal to H, reaction of compound of FormulaXV′ with ammonia in a suitable solvent, afforded compound of FormulaI-ACA′. Suitable solvents for use in the above process included, butwere not limited to, ethers such as tetrahydrofuran (THF), glyme, andthe like; dimethylformamide (DMF); dimethyl sulfoxide (DMSO);acetonitrile; alcohols such as methanol, ethanol, isopropanol,trifluoroethanol, and the like; and chlorinated solvents such asmethylene chloride (CH₂Cl₂) or chloroform (CHCl₃). If desired, mixturesof these solvents were used, however, the preferred solvent wasisopropanol. The above process was carried out at temperatures betweenabout −78° C. and about 120° C. Preferably, the reaction was carried outbetween 80° C. and about 100° C. The above process to produce compoundsof the present invention was preferably carried out in a glass pressuretube or a stainless steel reactor. Preferably, an excess of ammonia wasused. Additionally, in a typical preparation of compound of FormulaI-ACA′ (compounds of Formula I-ACA where R³═Z—CONR³¹²R³²²), compound ofFormula XV′ (compounds of Formula XV′ where R³═Z—CO₂A³) was reacted withHNR³¹²R³²² followed by ammonia in a suitable solvent. When A³=H, typicalcoupling procedures (such as conversion of —CO₂H to —COCl via treatmentwith SOCl₂ or oxalyl chloride followed by reaction with HNR³¹²R³²² ortreatment of —CO₂H and HNR³¹²R³²² with EDC or DCC in conjunction withDMAP, HOBT, or HOAt and the like) were employed to afford thetransformation of a carboxylic acid to an amide. When A³=alkyl such asmethyl or ethyl, treatment of the ester with Al(NR³¹²R³²²) affordedconversion of —CO₂A³ to —CO(NR³¹²R³²²). Subsequent treatment withammonia afforded compounds of Formula I-ACA′.

The chemistry shown in Scheme 34 can also be applied to compounds withQ¹ in place of A¹.

The compounds of Formula XVIII (compounds of Formula XV, I-ACA, or I-ACwhere R³═Z—CH₂OH), XIX (compounds of Formula XV, I-ACA, or I-AC whereR³═Z—CH₂LG), and XX (compounds of Formula XV, I-ACA, or I-AC whereR³═Z—CH₂A⁵(R³¹³)(R³²³)_(aa)) were prepared as shown below in Scheme 35:

where Q¹, R³¹³, and R³²³ are as defined previously for compound ofFormula I; LG=suitable leaving group such as tosylate, mesylate,trifluoromethanesulfonate, or halo such as chloro, bromo, or iodo; aa=0or 1; A³=hydrogen or alkyl such as methyl or ethyl; A¹¹=halogen such asCl, Br, or I; A¹²=C₁ or NH₂; A¹³=A¹¹ or Q¹; and A⁵=N, O or S.

The following table indicates the relations between the compounds ofFormulas XVII-XX, A¹², A¹³, compounds of Formulas I-AC, I-ACA, and XV,and R³.

Compound . . . is of wherein and equal to Formula. . . A¹² = A¹³ =Formula . . . wherein R³ = XVII Cl A¹¹ XV Z—CO₂A³ XVII NH₂ A¹¹ I-ACAZ—CO₂A³ XVII NH₂ Q¹ I-AC Z—CO₂A³ XVIII Cl A¹¹ XV Z—CH₂OH XVIII NH₂ A¹¹I-ACA Z—CH₂OH XVIII NH₂ Q¹ I-AC Z—CH₂OH XIX Cl A¹¹ XV Z—CH₂LG XIX NH₂A¹¹ I-ACA Z—CH₂LG XIX NH₂ Q¹ I-AC Z—CH₂LG XX Cl A¹¹ XV Z—CH₂A⁵R²(R⁴)_(d)XX NH₂ A¹¹ I-ACA Z—CH₂A⁵R²(R⁴)_(d) XX NH₂ Q¹ I-AC Z—CH₂A⁵R²(R⁴)_(d)

In a typical preparation of compound of Formula XVIII (compounds ofFormula XV, I-ACA, or I-AC, where R³═Z—CH₂OH), compound of Formula XVII(compounds of Formula XV, I-ACA, or I-AC, where R³═Z—CO₂A³) is treatedwith a suitable reducing agent, such as lithium aluminum hydride ordiisobutylaluminum hydride, in a suitable solvent, such as THF ormethylene chloride, to afford compound of Formula XVIII. In a typicalpreparation of compound of Formula XX (compounds of Formula XV, I-ACA,or I-AC, where R³═Z—CH₂A⁵(R³¹³)(R³²³)_(aa)), the hydroxy group ofcompound of Formula XVIII was converted to a suitable leaving group, LG,such as Cl or tosylate, mesylate, or triflate, by reaction with SOCl₂ orTs₂O, Ms₂O, or Tf₂O to afford compound of Formula XIX (compounds ofFormula XV, I-ACA, or I-AC, where R³═Z—CH₂LG). Reaction of compound ofFormula XIX with HA⁵(R³¹³)(R³²³)_(aa) afforded compound of Formula XX.Furthermore, compound of Formula XVIII can be directly converted tocompound of Formula XX by treating compound of Formula XVIII withvarious alkylating agents or under typical Mitsunobu reaction conditionsto afford compounds of Formula XX (compounds of Formula XV, I-ACA, orI-AC, where R³═Z—CH₂A⁵(R³¹³)(R³²³)_(aa)) in which A⁵=O, aa=0, andR³¹³=alkyl or aryl). Someone skilled in the art will choose the mostappropriate stage during the sequence shown in Scheme 35 to convertA¹²=Cl to A¹²=NH₂ as described in Scheme 31, and to convert A¹³=A¹ toA¹³=Q¹ as described in Scheme 30, if applicable.

An alternative preparation of compounds of Formula I-AC is shown inScheme 36.

where Q¹ and R³ are as defined previously for compound of Formula I; andA¹=halogen such as Cl, Br, or I.

The compounds of Formula XXI may be prepared from aldehydes Q¹-CHO (seeScheme 14 for their preparation) by addition of methyllithium or amethyl Grignard reagent, followed by oxidation of the resulting alcoholto the ketone of Formula XXI. Other compounds are commercially availableor can be prepared by methods well known to someone skilled in the art,see: Larock, R. C. Comprehensive Organic Transformations, 2^(nd) ed.;Wiley and Sons: New York, 1999, 1197ff. Reaction of compounds of FormulaXXI under typical halogenation conditions with typical halogenatingagents including, but not limited to, Br₂, NBS, pyridinium perbromide,or CuBr₂ (for A¹¹=Br), or NCS or SO₂Cl₂ (for A¹¹=Cl) gives the compoundsof Formula XXII. Their reaction with amines of Formula H₂N—R³ gives theaminoketones of Formula XXIII that are converted to aminocyanopyrrolesof Formula XXIV by reaction with malononitrile under basic conditions.Finally, reaction of compounds of Formula XXIV under typical cyclizationconditions gives the compounds of Formula I-AC. Conditions for thiscyclization include, but are not limited to, heating with formamide;heating with formamide and ammonia; sequential treatment with a trialkylorthoformate, ammonia, and a base; sequential treatment with formamidineand ammonia.

It would be appreciated by those skilled in the art that in somesituations, a substituent that is identical or has the same reactivityto a functional group which has been modified in one of the aboveprocesses, will have to undergo protection followed by deprotection toafford the desired product and avoid undesired side reactions.Alternatively, another of the processes described within this inventionmay be employed in order to avoid competing functional groups. Examplesof suitable protecting groups and methods for their addition and removalmay be found in the following reference: “Protective Groups in OrganicSyntheses”, T. W. Greene and P. G. M. Wuts, John Wiley and Sons, 1989.

Compound of Formula I-AQ is equal to compound of Formula I whereinX₁═CH; X₂, X₃ and X₅═N; X₄, X₆, and X₇═C and J=H or NH₂

Method AQ was used when preparing compounds of Formula I-AQ as shownbelow in Scheme 37:

Method AQ:

where Q¹ and R³ are as defined previously for compound of Formula I,A¹¹=halogen such as Cl, Br, or I; B(OR)₂=suitable boronic acid/ester andJ=H or NH₂.

In a typical preparation of compounds of Formula I-AQ, compound ofFormula II-Q was reacted with a suitable boronic acid/ester (Q¹-B(OR)₂)in a suitable solvent via typical Suzuki coupling procedures. Suitablesolvents for use in the above process included, but were not limited to,water, ethers such as tetrahydrofuran (THF), glyme, and the like;dimethylformamide (DMF); dimethyl sulfoxide (DMSO); acetonitrile;alcohols such as methanol, ethanol, isopropanol, trifluoroethanol, andthe like; and chlorinated solvents such as methylene chloride (CH₂Cl₂)or chloroform (CHCl₃). If desired, mixtures of these solvents were used,however, the preferred solvent was glyme/water. The above process wascarried out at temperatures between about −78° C. and about 120° C.Preferably, the reaction was carried out between 80° C. and about 100°C. The above process to produce compounds of the present invention waspreferably carried out at about atmospheric pressure although higher orlower pressures were used if desired. Substantially equimolar amounts ofreactants were preferably used although higher or lower amounts wereused if desired.

One skilled in the art will appreciate that alternative methods may beapplicable for preparing compounds of Formula I-AQ from II-Q. Forexample, compound of Formula II-Q could be reacted with a suitableorganotin reagent Q¹-SnBu₃ or the like in a suitable solvent via typicalStille coupling procedures.

The compounds of Formula II-Q of Scheme 37 were prepared as shown belowin Scheme 38.

where R³ is as defined previously for compound of Formula I andA¹¹=halogen such as Cl, Br, or I; and J=H or NH₂.

In a typical preparation of compounds of Formula II-Q, compound ofFormula III-Q was reacted with phosphorus oxychloride (POCl₃) andtriazole, and pyridine followed by ammonia (NH₃) in a suitable solvent.Suitable solvents for use in the above process included, but were notlimited to, ethers such as tetrahydrofuran (THF), glyme, and the like;dimethylformamide (DMF); dimethyl sulfoxide (DMSO); acetonitrile;alcohols such as methanol, ethanol, isopropanol, trifluoroethanol, andthe like; and chlorinated solvents such as methylene chloride (CH₂Cl₂)or chloroform (CHCl₃). If desired, mixtures of these solvents were used,however, the preferred solvent was isopropanol. The above process wascarried out at temperatures between about −20° C. and about 50° C.Preferably, the reaction was carried out between 0° C. and about 25° C.The above process to produce compounds of the present invention waspreferably carried out at about atmospheric pressure although higher orlower pressures were used if desired. Substantially equimolar amounts ofreactants were preferably used although higher or lower amounts wereused if desired.

The compounds of Formula III-Q of Scheme 38 were prepared as shown belowin Scheme 39.

where R³ is as defined previously for compound of Formula I; A¹¹=halogensuch as Cl, Br, or I; and J=H or NH₂.

In a typical preparation of a compound of Formula III-Q, intermediateV-Q was converted to compound of Formula IV-Q. Intermediate of FormulaV-Q was treated with phosphorus oxychloride (POCl₃) in a suitablesolvent at a suitable reaction temperature. Suitable solvents for use inthe above process included, but were not limited to, ethers such astetrahydrofuran (THF), glyme, and the like, chlorinated solvents such asmethylene chloride (CH₂Cl₂) or chloroform (CHCl₃), and acetonitrile. Ifdesired, mixtures of these solvents were used. The preferred solvent wasacetonitrile. The above process was carried out at temperatures betweenabout −78° C. and about 120° C. Preferably, the reaction was carried outbetween 40° C. and about 95° C. The above process to produce compoundsof the present invention was preferably carried out at about atmosphericpressure although higher or lower pressures were used if desired.Intermediate for Formula III-Q was prepared by reacting intermediate ofFormula IV-Q with a suitable halogenating agent. Suitable halogenatingagents included, but were not limited to, Br₂, I₂, Cl₂,N-chlorosuccinimide, N-bromosuccinimide, or N-iodosuccinimide. Thepreferred halogenating agent was N-iodosuccinimide. Suitable solventsfor use in the above process included, but were not limited to, etherssuch as tetrahydrofuran (THF), glyme, and the like; dimethylformamide(DMF); dimethyl sulfoxide (DMSO); acetonitrile; alcohols such asmethanol, ethanol, isopropanol, trifluoroethanol, and the like; andchlorinated solvents such as methylene chloride (CH₂Cl₂) or chloroform(CHCl₃). If desired, mixtures of these solvents were used, however, thepreferred solvent was DMF. The above process was carried out attemperatures between about −78° C. and about 120° C. Preferably, thereaction was carried out between 40° C. and about 75° C. The aboveprocess to produce compounds of the present invention was preferablycarried out at about atmospheric pressure although higher or lowerpressures were used if desired. Substantially equimolar amounts ofreactants were preferably used although higher or lower amounts wereused if desired.

Compounds of Formulae IV-Q and III-Q where J=NH₂ can be respectivelyconverted into the compounds of Formulae IV-Q and III-Q where J=H, bydiazotisation procedures known to those skilled in the art. A typicalprocedure includes the treatment of a compound of Formula IV-Q or III-Qwhere J=NH₂ with tert-butylnitrite in a suitable solvent such a THF orDMF.

The compounds of Formula V-Q of Scheme 39 were prepared as shown belowin Scheme 40:

where R¹ is as defined previously for compound of Formula I; A¹=OH,alkoxy, or a leaving group such as chloro or imidazole; and J=H or NH₂.

In a typical preparation, of a compound of Formula V-Q, a compound ofFormula VI-Q and compound of Formula V were reacted under suitableamide-coupling conditions. Suitable conditions include but are notlimited to treating compounds of Formula VI-Q and V (when A¹=OH) withcoupling reagents such as DCC or EDC in conjunction with DMAP, HOBt,HOAt and the like, or reagents like EEDQ. Suitable solvents for use inthe above process included, but were not limited to, ethers such astetrahydrofuran (THF), glyme, and the like; dimethylformamide (DMF);dimethyl sulfoxide (DMSO); acetonitrile; halogenated solvents such aschloroform or methylene chloride. If desired, mixtures of these solventswere used, however the preferred solvent was methylene chloride. Theabove process was carried out at temperatures between about 0° C. andabout 80° C. Preferably, the reaction was carried out at about 22° C.The above process to produce compounds of the present invention waspreferably carried out at about atmospheric pressure although higher orlower pressures were used if desired. Substantially equimolar amounts ofreactants were preferably used although higher or lower amounts wereused if desired. Alternatively, compounds of Formula VI-Q and V (whereA¹=F, Cl, Br, I) were reacted with bases such as triethylamine orethyldiisopropylamine and the like in conjunction with DMAP and thelike. Suitable solvents for use in this process included, but were notlimited to, ethers such as tetrahydrofuran (THF), glyme, and the like;dimethylformamide (DMF); dimethyl sulfoxide (DMSO); acetonitrile;pyridine; halogenated solvents such as chloroform or methylene chloride.If desired, mixtures of these solvents were used, however the preferredsolvent was DMF. The above process was carried out at temperaturesbetween about −20° C. and about 40° C. Preferably, the reaction wascarried out between 0° C. and 25° C. The above process to producecompounds of the present invention was preferably carried out at aboutatmospheric pressure although higher or lower pressures were used ifdesired. Substantially equimolar amounts of compounds of Formula VI-Qand V (where A¹=F, Cl, Br, I) and base and substoichiometric amounts ofDMAP were preferably used although higher or lower amounts were used ifdesired. Additionally, other suitable reaction conditions for theconversion of an amine (compound of Formula VI-Q) to an amide (compoundof Formula V-Q) can be found in Larock, R. C. Comprehensive OrganicTransformations, 2^(nd) ed.; Wiley and Sons: New York, 1999, pp1941-1949.

The compounds of Formula VI-Q of Scheme 40 where J=H were prepared asshown below in Scheme 41:

In a typical preparation, of a compound of Formula VI-Q, a compound ofFormula VII-Q is reacted under suitable reaction conditions in asuitable solvent. Suitable conditions include treatment of compound ofFormula VII-Q with hydrazine or methyl hydrazine in a suitable solvent.Suitable solvents for use in the above process included, but were notlimited to, ethers such as tetrahydrofuran (THF), glyme, and the like;dimethylformamide (DMF); dimethyl sulfoxide (DMSO); acetonitrile;halogenated solvents such as chloroform or methylene chloride; alcoholicsolvents such as methanol and ethanol. If desired, mixtures of thesesolvents may be used, however the preferred solvents were ethanol andmethylene chloride. The above process was carried out at temperaturesbetween about 0° C. and about 80° C. Preferably, the reaction wascarried out at about 22° C. The above process to produce compounds ofthe present invention was preferably carried out at about atmosphericpressure although higher or lower pressures were used if desired.Substantially equimolar amounts of reactants were preferably usedalthough higher or lower amounts were used if desired.

Compounds of Formula VI-Q where J=NH₂ may be prepared according to theprocedures described in J. Het. Chem., (1984), 21, 697.

The compounds of Formula VII-Q of Scheme 41 were prepared as shown belowin Scheme 42:

In a typical preparation of a compound of Formula VII-Q, a compound ofFormula VIII-Q was reacted with Raney Nickel in a suitable solvent.Suitable solvents for use in the above process included, but were notlimited to, ethers such as tetrahydrofuran (THF), glyme, and the like;dimethylformamide (DMF); dimethyl sulfoxide (DMSO); acetonitrile(CH₃CN); alcohols such as methanol, ethanol, isopropanol,trifluoroethanol, and the like; chlorinated solvents such as methylenechloride (CH₂Cl₂) or chloroform (CHCl₃). If desired, mixtures of thesesolvents were used, however, the preferred solvent was ethanol. Theabove process may be carried out at temperatures between about rt andabout 100° C. Preferably, the reaction was carried out at about 80° C.The above process to produce compounds of the present invention waspreferably carried out at about atmospheric pressure although higher orlower pressures were used if desired. Substantially equimolar amounts ofreactants were preferably used although higher or lower amounts wereused if desired. Additionally a compound of Formula VII-Q can beprepared by reacting a compound of Formula VIII-Q with a suitableoxidizing agent in a suitable solvent. A suitable oxidizing agentincludes, but is not limited to hydrogen peroxide (H₂O₂), 3-chloroperoxybenzoic acid (mCPBA) and the like. Suitable solvents for use inthe above process included, but were not limited to, ethers such as THF,glyme, and the like; DMF; DMSO; CH₃CN; and dimethylacetamide (DMA);chlorinated solvents such as CH₂Cl₂ or CHCl₃ If desired, mixtures ofthese solvents were used, however, the preferred solvent was DMA. Theabove process may be carried out at temperatures between about 0° C. and100° C. Preferably, the reaction was carried out at about rt to 70° C.The above process to produce compounds of the present invention waspreferably carried out at about atmospheric pressure although higher orlower pressures were used if desired. Substantially equimolar amounts ofreactants were preferably used although higher or lower amounts wereused if desired.

The compounds of Formula VIII-Q of Scheme 42 were prepared as shownbelow in Scheme 43:

In a typical preparation of a compound of Formula VIII-Q, a compound ofFormula IX-Q was reacted with thiosemicarbazide and a suitable base in asuitable solvent. Suitable bases include, but were not limited totriethylamine, ethyldiisopropylamine and the like. Suitable solvents foruse in the above process included, but were not limited to, ethers suchas tetrahydrofuran (THF), glyme, and the like; dimethylformamide (DMF);dimethylacetamide (DMA); dimethyl sulfoxide (DMSO); acetonitrile(CH₃CN); alcohols such as methanol, ethanol, isopropanol,trifluoroethanol, and the like; chlorinated solvents such as methylenechloride (CH₂Cl₂) or chloroform (CHCl₃). If desired, mixtures of thesesolvents were used, however, the preferred solvent was ethanol. Theabove process may be carried out at temperatures between about rt andabout 100° C. Preferably, the reaction was carried out between about 40°C. and 80° C. The above process to produce compounds of the presentinvention was preferably carried out at about atmospheric pressurealthough higher or lower pressures were used if desired. Substantiallyequimolar amounts of reactants were preferably used although higher orlower amounts were used if desired. Compound of Formula IX-Q can beprepared according to literature procedures Knutsen, Lars J. S. et. al.,J. Chem. Soc. Perkin Trans 1: Organic and Bio-Organic Chemistry(1972-1999), 1984, 229-238.

It would be appreciated by those skilled in the art that in somesituations, a substituent that is identical or has the same reactivityto a functional group which has been modified in one of the aboveprocesses, will have to undergo protection followed by deprotection toafford the desired product and avoid undesired side reactions.Alternatively, another of the processes described within this inventionmay be employed in order to avoid competing functional groups. Examplesof suitable protecting groups and methods for their addition and removalmay be found in the following reference: “Protective Groups in OrganicSyntheses”, T. W. Greene and P. G. M. Wuts, John Wiley and Sons, 1989.

Method AW was also used when preparing compounds of Formula II-Q asshown below in Scheme 44:

Method AW:

where Q¹ and R³ are as defined previously for compound of Formula I, andA¹¹=halogen such as Cl, Br, or I.

In a typical preparation of compounds of Formula II-Q, compound ofFormula III-W was reacted with ammonia in a suitable solvent. Suitablesolvents for use in the above process included, but were not limited to,ethers such as tetrahydrofuran (THF), glyme, and the like; alcohols suchas methanol, ethanol, isopropanol, trifluoroethanol, and the like; andchlorinated solvents such as methylene chloride (CH₂Cl₂) or chloroform(CHCl₃). If desired, mixtures of these solvents were used, however, thepreferred solvent was isopropanol. The above process was carried out attemperatures between about 0° C. and about 50° C. Preferably, thereaction was carried out at between 0° C. and about 22° C. The aboveprocess to produce compounds of the present invention was preferablycarried out at about atmospheric pressure although higher or lowerpressures were used if desired. Substantially equimolar amounts ofreactants were preferably used although higher or lower amounts wereused if desired.

The compounds of Formula III-W of Scheme 44 were prepared as shown belowin Scheme 45.

where R³ is as defined previously for compound of Formula I andA¹¹=halogen such as Cl, Br, or I.

In a typical preparation of a compound of Formula III-W, compound V-Wwas converted to compound of Formula IV-W. Compound of Formula V-W wastreated with phosphorus oxychloride (POCl₃) or the isolated “Vilsmeirsalt” [CAS#33842-02-3] in a suitable solvent at a suitable reactiontemperature. Suitable solvents for use in the above process included,but were not limited to, ethers such as tetrahydrofuran (THF), glyme,and the like, chlorinated solvents such as methylene chloride (CH₂Cl₂)or chloroform (CHCl₃), and acetonitrile (CH₃CN). If desired, mixtures ofthese solvents were used. The preferred solvent was acetonitrile. Theabove process was carried out at temperatures between about −78° C. andabout 120° C. Preferably, the reaction was carried out between 40° C.and about 95° C. The above process to produce compounds of the presentinvention was preferably carried out at about atmospheric pressurealthough higher or lower pressures were used if desired. Compounds ofFormula III-W were prepared by reacting compound of Formula IV-W with asuitable halogenating agent. Suitable halogenating agents included, butwere not limited to, Br₂, I₂, Cl₂, N-chlorosuccinimide,N-bromosuccinimide, or N-iodosuccinimide. The preferred halogenatingagent was N-iodosuccinimide. Suitable solvents for use in the aboveprocess included, but were not limited to, ethers such astetrahydrofuran (THF), glyme, and the like; dimethylformamide (DMF);dimethyl sulfoxide (DMSO); acetonitrile; alcohols such as methanol,ethanol, isopropanol, trifluoroethanol, and the like; and chlorinatedsolvents such as methylene chloride (CH₂Cl₂) or chloroform (CHCl₃). Ifdesired, mixtures of these solvents were used, however, the preferredsolvent was DMF. The above process was carried out at temperaturesbetween about −78° C. and about 120° C. Preferably, the reaction wascarried out between 40° C. and about 75° C. The above process to producecompounds of the present invention was preferably carried out at aboutatmospheric pressure although higher or lower pressures were used ifdesired. Substantially equimolar amounts of reactants were preferablyused although higher or lower amounts were used if desired.

The compounds of Formula V-W of Scheme 45 were prepared as shown belowin Scheme 46.

where R³ is as defined previously for compound of Formula I, X¹²=azido,or mono- or di-protected amino and A¹=OH, alkoxy or a leaving group suchas chloro or imidazole.

In a typical preparation of a compound of Formula V-W, compound VI-W wasreacted with compound V under suitable amide coupling conditions.Suitable conditions include but are not limited to those described forthe conversion of compound XIII to compound XII as shown in Scheme 10.Compounds of Formula VI-W were prepared from compounds of Formula VII-W.A typical procedure for the conversion of compounds of Formula VII-W tocompounds of Formula VI-W involves subjecting a compound of FormulaVII-W, where X¹²=azido, to reducing conditions such as, but not limitedto, catalytic hydrogenation in a suitable solvent at a suitable reactiontemperature. Suitable solvents for use in the above process included,but were not limited to, ethers such as tetrahydrofuran (THF), glyme,and the like, alcoholic solvents such as methanol, ethanol and the like,esters such as ethyl acetate, methyl acetate and the like. If desired,mixtures of these solvents were used. The preferred solvents were ethylacetate and methanol. The above process was carried out at temperaturesbetween about −78° C. and about 120° C. Preferably, the reaction wascarried out between 40° C. and about 95° C. The above process to producecompounds of the present invention was preferably carried out at aboutatmospheric pressure although higher or lower pressures were used ifdesired. Alternatively, when X¹²=azido, the reduction to compounds ofFormula VI-W could be achieved by treatment of a compound of FormulaVII-W with triaryl- or trialkylphosphines in the presence of water in asuitable solvent at a suitable reaction temperature. Suitable solventsfor use in the above process included, but were not limited to, etherssuch as tetrahydrofuran (THF), dioxane and the like, alcoholic solventssuch as methanol, ethanol and the like, esters such as ethyl acetate,methyl acetate and the like, DMF, acetonitrile, and pyridine. Ifdesired, mixtures of these solvents were used. The preferred solventswere THF and acetonitrile. The above process was carried out attemperatures between about −78° C. and about 120° C. Preferably, thereaction was carried out between 40° C. and about 95° C. The aboveprocess to produce compounds of the present invention was preferablycarried out at about atmospheric pressure although higher or lowerpressures were used if desired.

Where X¹²=mono- or di-protected amino, the deprotection could beeffected by the procedures known to those skilled in the art and asdisclosed in: “Protective Groups in Organic Syntheses”, T. W. Greene andP. G. M. Wuts, John Wiley and Sons, 1989.

The compounds of Formula VII-W of Scheme 46 were prepared as shown belowin Scheme 47:

where R₃ is as defined previously for compound of Formula I, X¹² is asdefined for a compound of Formula VII-W and A¹²=iodo, bromo, chloro,tosylate, mesylate or other leaving group.

In a typical preparation of a compound of Formula VII-W where X¹²=azide,compound VIII-W was reacted with an azide salt, such as lithium orsodium azide in suitable solvent at a suitable reaction temperature.Suitable solvents for use in the above process included, but were notlimited to, alcoholic solvents such as ethanol, butanol and the like,esters such as ethyl acetate, methyl acetate and the like, DMF,acetonitrile, acetone DMSO. If desired, mixtures of these solvents wereused. The preferred solvents were acetone and DMF. The above process wascarried out at temperatures between about −78° C. and about 120° C.Preferably, the reaction was carried out between 40° C. and about 95° C.The above process to produce compounds of the present invention waspreferably carried out at about atmospheric pressure although higher orlower pressures were used if desired. Alternatively, where X¹²=mono- ordi-protected amino, compounds of Formula VIII-W were reacted withsuitably protected amines where the protecting group is chosen such thatthe nucleophilic nature of the nitrogen is either retained or where itcan be enhanced by the action of a reagent such as a base. Those skilledin the art will recognize that such protecting groups include, but arenot limited to, benzyl, trityl, allyl, and alkyloxycarbonyl derivativessuch as BOC, CBZ and FMOC.

Compounds of Formula VIII-W where A¹²=halogen, are prepared fromcompounds of Formula XI-W. In a typical procedure, compounds of FormulaXI-W are treated with halogenating reagents such as but not limited toN-iodosuccinimide, N-bromosuccinimide, N-chlorosuccinimide,trichloroisocyanuric acid, N,N′-1,3-dibromo-5,5-dimethylhydantoin,bromine and iodine, preferably in the presence of one or more radicalsources such as dibenzoyl peroxide, azobisisobutyronitrile or light insuitable solvent at a suitable reaction temperature. Suitable solventsfor use in the above process included, but were not limited to,chlorinated solvents such as carbon tetrachloride, dichloromethane,α,α,α-trifluorotoluene and the like, esters such as methyl formate,methyl acetate and the like, DMF, acetonitrile. If desired, mixtures ofthese solvents were used. The preferred solvents were carbontetrachloride and α,α,α-trifluorotoluene. The above process was carriedout at temperatures between about −78° C. and about 120° C. Preferably,the reaction was carried out between 40° C. and about 95° C. The aboveprocess to produce compounds of the present invention was preferablycarried out at about atmospheric pressure although higher or lowerpressures were used if desired.

Alternatively, compounds of Formula VIII-W where A¹²=tosylate ormesylate were prepared from compounds of Formula X-W as shown in Scheme48. In a typical preparation of a compound of Formula VIII-W, a compoundof Formula X-W was reacted with a sulfonylating reagent such asmethanesulfonyl chloride or p-toluenesulfonyl chloride in the presenceof a base such as, but not limited to DIPEA or triethylamine in asuitable solvent at a suitable reaction temperature. Suitable solventsfor use in the above reaction included, but were not limited to,chlorinated solvents such as dichloromethane, 1,2□-dichloroethane andthe like, ethers such THF, diethylether and the like, DMF andacetonitrile. If desired, mixtures of these solvents were used. Thepreferred solvents were THF and dichloromethane. The above process wascarried out at temperatures between about −78° C. and about 120° C.Preferably, the reaction was carried out between 40° C. and about 95° C.The above process to produce compounds of the present invention waspreferably carried out at about atmospheric pressure although higher orlower pressures were used if desired.

Compounds of Formula X-W were prepared from compounds of Formula XI-W.In a typical preparation of a compound of Formula X-W, a compound ofFormula XI-W was reacted with a reducing reagent such as, but notlimited to, sodium borohydride, lithium borohydride or lithium aluminumhydride in a suitable solvent at a suitable reaction temperature.Suitable solvents for use in the above reaction included, but were notlimited to, ethers such THF, diethylether and the like, and alcoholssuch as ethanol, methanol, isopropanol and the like. If desired,mixtures of these solvents were used. The preferred solvents were THFand methanol. The above process was carried out at temperatures betweenabout −78° C. and about 120° C. Preferably, the reaction was carried outbetween 40° C. and about 95° C. The above process to produce compoundsof the present invention was preferably carried out at about atmosphericpressure although higher or lower pressures were used if desired.

Compounds of Formula XI-W were prepared from compounds of Formula XI-W.In a typical preparation of a compound of Formula XI-W, a compound ofFormula IX-W was reacted with an oxidizing reagent such as, but notlimited to, selenium dioxide, manganese dioxide, potassium permanganateand the like, in a suitable solvent at a suitable reaction temperature.Suitable solvents for use in the above reaction included, but were notlimited to, chlorinated solvents such as dichloromethane,1,2-dichloroethane and the like, water, acetic acid and sulfolane. Ifdesired, mixtures of these solvents were used. The above process wascarried out at temperatures between about −78° C. and about 120° C.Preferably, the reaction was carried out between 40° C. and about 95° C.The above process to produce compounds of the present invention waspreferably carried out at about atmospheric pressure although higher orlower pressures were used if desired.

Those skilled in the art will appreciate that compounds of Formula IX-Wcan be made by routes disclosed in the literature, for example as inBulletin de la Societe Chimique de France, (1973), (6) (Pt. 2), 2126.

Compounds of Formula I-AQ and/or their precursors may be subjected tovarious functional group interconversions as a means to access somefunctionalities that may not be introduced directly as a result ofincompatible chemistries. Examples of such functional groupmanipulations applicable to compounds of Formula I-AQ and theirprecursors are similar, but not limited to, those described in Schemes16-27, 34 and 35 that related to compounds of Formula I-AA, I-P, I-P′,I-Q, I-R, I-AB and I-AC.

EXPERIMENTAL PROCEDURES 8-Chloro-3-cyclobutyl-imidazo[1,5-a]pyrazine

This compound was prepared using procedures analogous to that describedfor trans-methyl4-(8-chloroimidazo[1,5-a]pyrazin-3-yl)cyclohexanecarboxylate and itsprecursor trans-methyl4-({[(3-chloropyrazin-2-yl)methyl]amino}carbonyl)cyclohexanecarboxylate,using cyclobutanecarboxylic acid in place of4-(methoxycarbonyl)cyclohexanecarboxylic acid.

8-Chloro-3-cyclobutyl-1-iodoimidazo[1,5-a]pyrazine

8-Chloro-3-cyclobutylimidazo[1,5-a]pyrazine (1058 mg, 5.1 mmol) and NIS(1146 mg, 5.1 mmol) in anh DMF (10 mL) were stirred at 60° C. under Arfor 6 h. The reaction was diluted with DCM (˜400 mL), washed (H₂O,brine), dried (Na₂SO₄) and concentrated under reduced pressure.Purification of the crude material by flash chromatography on silica gel(50 g cartridge, 10:1-8:1-7:1-6:1 hexanes:EtOAc) afforded the titlecompound as a pale yellow solid; ¹H NMR (400 MHz, CDCl₃) δ 7.51 (d,J=4.8 Hz, 1H), 7.26 (d, J=4.8 Hz, 1H), 3.75 (quintetd, J=1.2 Hz, 8.4 Hz,1H), 2.62-2.42 (m, 4H), 2.32-1.98 (m, 2H); MS (ES+): m/z 334.0 (100)[MH⁺]; HPLC: t_(R)=3.38 min (OpenLynx, polar_(—)5 min).

3-Cyclobutyl-1-iodoimidazo[1,5-a]pyrazin-8-amine

A Parr bomb containing8-chloro-3-cyclobutyl-1-iodoimidazo[1,5-a]pyrazine (759 mg, 2.3 mmol) inIPA (100 mL) was saturated with NH₃(g) for 5 min at 0° C. then sealedand heated at 115° C. for 38 h. The reaction mixture was thenconcentrated under reduced pressure, partitioned between DCM (200 mL)and H₂O (50 mL) and extracted with DCM (50 mL). Combined organicfractions were washed with brine, dried (Na₂SO₄) and concentrated underreduced pressure to provide the title compound as a white solid; ¹H NMR(400 MHz, CDCl₃) δ 7.13 (d, J=4.8 Hz, 1H), 7.01 (d, J=5.2 Hz, 1H), 5.63(br, 2H), 3.73 (quintetd, J=0.8 Hz, 8.4 Hz, 1H), 2.60-2.38 (m, 4H),2.20-1.90 (m, 2H); MS (ES+): m/z 315.9 (100) [MH⁺]; HPLC: t_(R)=1.75 min(OpenLynx, polar_(—)5 min).

7-Cyclohexyl-5-iodoimidazo[5,1-f][1,2,4]triazin-4-amine

To a suspension of 1H-1,2,4-triazole (1 g, 0.02 mol) in acetonitrile (23mL) was added dropwise phosphoryl chloride (0.6 mL, 0.007 mol) andtriethylamine (3 mL, 0.02 mol) at 0° C. To this mixture was added7-cyclohexyl-5-iodoimidazo[5,1-f][1,2,4]triazin-4(3H)-one (77 mg, 0.224mmol) and the resulting mixture refluxed overnight. The cooled mixturewas then quenched with excess NH₃ in ^(i)PrOH (pH 8) stirred at rt for30 min. then filtered and the isolated solid washed with DCM. Thefiltrate was concentrated in vacuo and purified by chromatography oversilica gel eluting with 2% MeOH in DCM to afford the7-cyclohexyl-5-iodoimidazo[5,1-f][1,2,4]triazin-4-amine. ¹H NMR (400MHz-DMSO-d6) δ 1.14-1.91 (m, 10H), 3.11-3.18 (m, 1H), 6.75 (br.s, 1H),7.84 (s, 1H) 8.42 (bs, 1H); MS (ES+): m/z: 344.01 (100) [MH+]. HPLC:t_(R)=3.10 min (OpenLynx: polar_(—)5 min).

7-Cyclohexyl-5-iodoimidazo[5,1-f][1,2,4]triazin-4(3H)-one

To a solution of 7-cyclohexylimidazo[5,1-f][1,2,4]triazin-4(3H)-one (130mg, 0.6 mmol) in DMF (0.6 mL) was added N-iodosuccinimide (700 mg, 0.003mol) and the reaction mixture stirred at 55° C. for 20 h. After thistime the mixture was diluted with water (50 mL) and extracted with EtOAc(4×40 mL). The organic extracts were washed with water (4×40 mL),treated with sodium thiosulfate and brine, dried over Na₂SO₄ andconcentrated in vacuo to afford7-cyclohexyl-5-iodoimidazo[5,1-f][1,2,4]triazin-4(3H)-one. ¹H NMR (400MHz-DMSO-d6) δ 1.34-1.37 (m, 3H), 1.52-1.56 (m, 2H), 1.76-1.88 (m, 5H),3.06-3.08 (m, 1H) 7.87 (s, 1H) 11.78 (s, 1H); MS (ES+): m/z: 344.95(100) [MH+]. HPLC: t_(r)=2.95 min (OpenLynx: polar_(—)5 min).

7-Cyclohexylimidazo[5,1-f][1,2,4]triazin-4(3H)-one

To a suspension of 6-aminomethyl-4H-[1,2,4]triazin-5-one (250 mg, 1.98mmol) in DMF (7.5 mL) was added2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate(760 mg, 2.38 mmol), cyclohexanecarboxylic acid (305 mg, 2.38 mmol) andN,N-diisopropylethylamine (1.5 mL, 8.6 mmol). After 1 h acetonitrile (40mL) was added to the mixture followed by dropwise addition of phosphorylchloride (0.28 mL, 3.0 mmol) and the reaction mixture stirred at 55° C.for 1 h. The mixture was then concentrated in vacuo chromatographed oversilica gel eluting with 3% MeOH in DCM, to afford7-cyclohexylimidazo[5,1-f][1,2,4]triazin-4(3H)-one. ¹H NMR (400MHz-DMSO-d6) δ 1.24-1.91 (m, 10H), 3.08-3.16 (m, 1H), 7.68 (s, 1H) 7.88(s, 1H) 11.76 (s, 1H); MS (ES+): m/z: 219.24 (100) [MH+]. HPLC:t_(R)=2.44 min (OpenLynx: polar_(—)5 min).

trans-[4-(8-Amino-1-iodoimidazo[1,5-a]pyrazin-3-yl)cyclohexyl]methanol

trans-[4-(8-chloro-1-iodoimidazo[1,5-a]pyrazin-3-yl)cyclohexyl]methanol(26.50 g, 67.66 mmol) was charged in a 400 mL steel bomb and wasdissolved in 2M NH₃ in isopropanol (300 mL) and anhydrous THF (10 mL).The reaction mixture was cooled to −78° C. Ammonia gas was bubbledvigorously into the solution for 8 min; then the bomb was tightly sealedand heated to 120° C. for 20 h. The crude reaction mixture wasconcentrated in vacuo, then the reaction residue was taken up withMeOH/CHCl₃, loaded onto silica gel. The mixture was purified by a silicagel glass column chromatography [eluted with 1:1 CH₂Cl₂/EtOAc to 10%˜7 NNH₃ in MeOH/CHCl₃] to afford the desired product as a beige cream whitesolid; MS (ES+): m/z 373.01 (100) [MH⁺], 373.98 (50) [MH⁺2]; t_(R)(polar-5 min/openlynx) 1.57 min.

trans-[4-(8-Chloro-1-iodoimidazo[1,5-a]pyrazin-3-yl)cyclohexyl]methanol

trans-[4-(8-Chloroimidazo[1,5-a]pyrazin-3-yl)cyclohexyl]methanol (18.00g, 67.74 mmol) and N-iodosuccinimide (19.81 g, 88.06 mmol) in anhydrousDMF (360 mL) were stirred at 60° C. under N₂ for 6 h. The reaction wasdiluted with DCM (˜600 mL), washed with water and brine, dried overanhydrous Na₂SO₄ and then concentrated in vacuo. The crude material waspurified by a silica gel flash chromatography (eluted with 1:2 EtOAc/DCMto 1:1 EtOAc/DCM) to obtain the desired product as a pale yellow solid;By ¹H NMR analysis, the product was contaminated with 0.35 eq. ofNIS-impurity. The product was carried onto the next reaction withoutfurther purification; MS (ES+): m/z 391.92 (100) [MH⁺], 393.88 (50)[MH⁺2], 394.89 (10) [MH⁺3]; t_(R) (polar-5 min/openlynx) 2.79 min.

trans-[4-(8-Chloroimidazo[1,5-a]pyrazin-3-yl)cyclohexyl]methanol

A THF solution (1.00 L) of trans-methyl4-(8-chloroimidazo[1,5-a]pyrazin-3-yl)cyclohexanecarboxylate (29.70 g,101.1 mmol) was cooled to −78° C. and was charged with LAH (1M in THF,25.3 mmol, 25.3 mL) dropwise. After 30 min., the reaction mixture wascharged with additional LAH (25.3 mmol) at −78° C. and then, allowed tostir at −78° C. for 1.5 h. The reaction was slowly warmed up to rt andstirred for additional 30 min. Ethyl acetate, Na₂SO₄.10H₂O, and silicagel were added to the reaction mixture and concentrated in vacuo to givean orange solid. The crude mixture was purified by a silica gel glasscolumn chromatography (eluted with 2:3 EtOAc/DCM to 100% EtOAc) toobtain the title compound as a slightly yellow-tinted white solid; ¹HNMR (CDCl₃, 400 MHz) δ 1.14-1.30 (m, 2H), 1.61-1.75 (m_(c), 1H), 1.84(ddd, J=13.2, 13.2, 13.2, 3.2 Hz, 2H), 1.98-2.13 (m, 4H), 2.19 (s, br,—OH), 2.94 (tt, J=11.6, 3.2 Hz, 1H), 3.56 (d, J=6.0 Hz, 2H), 7.31 (d,J=5.2 Hz, 1H), 7.64 (dd, J=5.2, 1.2 Hz, 1H), 7.79 (d, J=0.8 Hz, 1H); MS(ES+): m/z 266.21/268.17 (100/89) [MH⁺]. HPLC: t_(R)=2.38 min (OpenLynx,polar_(—)5 min). MS (ES+): m/z 266.21 (100) [MH⁺], 268.17 (80) [MH⁺2},289.18 (20) [MH⁺3]; t_(R) (polar-5 min/openlynx) 2.36 min.

General Procedure for the Hydrolysis of Carboxylic Esters

To a solution/slurry of the carboxylic ester (30.17 mmol) in ethanol(200 mL) was added 3.0 M of sodium hydroxide in water (15.1 mL) and themixture was stirred at 40° C. for 4 h. The solvent was removed underreduced pressure at 40° C. and to the residue was added water (10 mL)and ethanol (10 mL) and the slurry was filtered.

The filter cake was washed with ethanol (2×10 mL) and dried under vacuumto yield the sodium salt. For the isolation of the free acid, water wasadded to this salt and the slurry was acidified with formic acid,stirred for 10 min at RT and filtered. The filter cake was washed withwater followed by ethanol to yield the carboxylic acid.

trans-Methyl4-(8-chloroimidazo[1,5-a]pyrazin-3-yl)cyclohexanecarboxylate

trans-Methyl4-({[(3-chloropyrazin-2-yl)methyl]amino}carbonyl)-cyclohexanecarboxylate(29.00 g, 93.02 mmol) was dissolved in anhydrous acetonitrile (930 mL)and anhydrous DMF (9 mL) and heated at 55° C. under nitrogen for 3 h.The reaction mixture was concentrated in vacuo, then, the solid residuewas taken up in DCM, then, basified to pH 10 with 2M ammonia inisopropanol. The mixture was concentrated in vacuo, re-dissolved in DCM,and then loaded onto TEA-basified silica gel. The crude product waspurified by a silica gel column chromatography (eluted with 2:3EtOAc/DCM) to obtain the title compound as a yellow powder; ¹H NMR(CDCl₃, 400 MHz) δ 1.63 (ddd, J=13.2, 13.2, 13.2, 3.2 Hz, 2H), 1.85(ddd, J=13.2, 13.2, 13.2, 2.8 Hz, 2H), 2.10 (dd, J=14.4, 3.2 Hz, 2H),2.19 (dd, J=14.0, 3.2 Hz, 2H), 2.46 (tt, J=12.4, 3.6 Hz, 1H), 2.96 (tt,J=11.6, 3.2 Hz, 1H), 3.70 (s, 3H), 7.33 (dd, J=5.2, 1.2 Hz, 1H), 7.61(d, J=4.8 Hz, 1H), 7.79 (s, 1H). MS (ES+): m/z 294.17/296.14 (100/86)[MH⁺]. HPLC: t_(R)=2.85 min (OpenLynx, polar_(—)5 min).

trans-Methyl4-({[(3-chloropyrazin-2-yl)methyl]amino}carbonyl)cyclohexanecarboxylate

A THF (370 mL) solution of 4-(methoxycarbonyl)cyclohexanecarboxylic acid(15.14 g, 81.30 mmol) and CDI (13.18 g, 81.30 mmol) was placed under anitrogen atmosphere and stirred at 60° C. for 4 h. The reaction mixturewas cooled to rt, then, (3-chloropyrazin-2-yl)methylaminebis-hydrochloride salt (16.00 g, 73.91 mmol) and DIPEA (31.52 g, 244.00mmol, 42.5 mL) was added. After stirring at 60° C. for 20 h, thereaction was concentrated in vacuo. The crude reaction mixture waspurified by a silica gel glass column chromatography (eluted with 3:2DCM/EtOAc) to obtain the pure desired product as a slightly yellowishcreamy white powder; ¹H NMR (CDCl₃, 400 MHz) δ 1.43-1.65 (m, 4H),2.01-2.14 (m, 4H), 2.25 (tt, J=12.0, 3.6 Hz, 1H), 2.34 (tt, J=11.6, 3.2Hz, 1H), 3.68 (s, 3H), 4.70 (d, J=4.4 Hz, 2H), 6.81 (s, br, —NH),8.32-8.36 (m, 1H), 8.46 (d, J=2.4 Hz, 1H); MS (ES+): m/z 312.17/314.12(84/32) [MH⁺]; HPLC: t_(R)=2.44 min (OpenLynx, polar_(—)5 min).

[3-(8-Amino-1-iodoimidazo[1,5-a]pyrazin-3-yl)-cyclobutyl]methanol

[3-(8-Chloro-1-iodoimidazo[1,5-a]pyrazin-3-yl)cyclobutyl]methanol (6.9g) in i-PrOH (200 mL) was saturated with NH₃(g), by passing a slow aslow stream of ammonia for 10 min at −20° C., and then heated in a Parrbomb at 110° C. for 2d. The reaction mixture was then cooled to rt,filtered through a sintered glass and the solid residue and the Parrvessel were rinsed with i-PrOH several times. The filtrate wasconcentrated under reduced pressure to provide an orange solid stillcontaining NH₄Cl. The material was taken up into refluxing MeCN (250 mL)and filtered hot. The step was repeated with another portion of hot MeCN(200 mL). The combined MeCN filtrates were concentrated under reducedpressure to give the title compound as an orange solid; HPLC: (polar5min) 0.53 and 1.51 min; MS (ES+): 345.1 (100, M⁺+1); ¹H NMR (400 MHz,DMSO-d6) δ 7.50 (d, J=5.2 Hz, 1H), 7.44 (d, J=5.2 Hz, 0.27H, minorisomer), 6.95 (d, J=5.2 Hz, 1.29H overlapped with the minor isomer) 6.63(br, 2H), 4.61 (t, J=5.2 Hz, 0.27H, minor isomer), 4.52 (t, J=5.2 Hz,1H), 3.69 (quintet, J=5.6 Hz, 0.32H, minor isomer), 3.54 (quintet, J=5.6Hz, 1H), 2.52-2.25 (m, 4H), 2.10-2.00 (m, 1H).

[3-(8-Chloro-1-iodo-imidazo[1,5-a]pyrazin-3-yl)-cyclobutyl]-methanol

To a solution of NIS (6.31 g, 28.0 mmol) in anh DMF (100 mL) under Arwas added dry [3-(8-chloroimidazo[1,5-a]pyrazin-3-yl)cyclobutyl]methanol(6.67 g) dissolved in anh DMF (30 mL). The flask containing[3-(8-chloroimidazo[1,5-a]pyrazin-3-yl)cyclobutyl]methanol was rinsedwith another portion of anh DMF (20 mL) and the rinse was added to thereaction mixture. The reaction was heated to 60° C. (rt→60° C.˜30 min)and the stirred at this temperature for 3 h. The mixture was then cooledto rt, partitioned between 1M aq Na₂S₂O₃ (60 mL), brine (60 mL) and DCM(160 mL). The aq layer was extracted with DCM (3×100 mL). The combinedorganics were dried (Na₂SO₄), concentrated under reduced pressure andpurified by flash chromatography on SiO₂ (0-8% MeOH in DCM) to provide amaterial, homogenous by UV on both TLC and HPLC, still containing DMF.The material was dissolved in DCM (200 mL) and washed with water (3×40mL), dried (Na₂SO₄) and concentrated under reduced pressure to providethe title compound as a pale yellow solid; HPLC (polar5 min) 2.52 min;MS (ES+): m/z (rel. int.) 364.0 (100, M⁺+1); ¹H NMR (400 MHz, CDCl₃) δ7.59 (d, J=4.8 Hz, 1H), 7.49 (d, J=4.8 Hz, 0.22H, minor isomer), 7.29(d, J=4.8 Hz, 1H), 7.28 (d, J=5.2 Hz, 0.23H, minor isomer), 3.83-3.80(m, 0.7H), 3.72-3.62 (m, 3H), 2.75-2.55 (m, 4H), 2.42-2.32 (m, 1-2H).

[3-(8-Chloro-imidazo[1,5-a]pyrazin-3-yl)-cyclobutyl]-methanol

To a solution of8-chloro-3-(3-methylenecyclobutyl)imidazo[1,5-a]pyrazine (4.48 g, 20.4mmol) in anh THF (255 mL) at −78° C. under Ar, 9-BBN (61.2 mL, 0.5M inTHF, 30.6 mmol) was added dropwise over 8 min (a suspension). Thecooling bath was replaced with ice-H₂O and the reaction was allowed towarm slowly to rt. After being stirred for 17 h, H₂O (100 mL,) was addedfollowed by, after ˜5 min, NaBO₃.H₂O (12.2 g, 122.3 mmol) added in onelot. The reaction was stirred at rt for 5 h and then filtered throughCelite. The Celite and residual solids were washed with DCM and EtOAc.The filtrate was concentrated under reduced pressure to yield an aqsolution, which was saturated with NaCl and extracted with EtOAc (3×).The extracts were dried (Na₂SO₄) and concentrated under reduced pressureto yield a light yellow oil which was purified by flash chromatographyon SiO₂ (9:1 DCM:MeOH) to afford the title compound as a light yellowoil; HPLC: t_(R) (mass-directed HPLC, polar7 min) 2.52 min; MS (ES+):238.0. The addition may be carried out at 0° C. Suspension quicklyclears up after the exchange of cooling baths. The final productcontained 1,5-cis-octanediol derived from 9-BBN. Based on ¹H NMRestimated roughly to be 66% target material and 33% of the byproduct.The crude product was taken onto next step crude, stereoselectivity ofthe product was 4-5:1 as judged by ¹H NMR.

(8-Chloro-3-(3-methylene-cyclobutyl)-imidazo[1,5a]pyrazine)

3-Methylene-cyclobutanecarboxylic acid(3-chloro-pyrazin-2-ylmethyl)-amide (52.1 g, 219.2 mmol) was dissolvedin 1.0 L of anhydrous MeCN. Followed by the addition of DMF (1.0 mL) andPOCl₃ (100 mL, 1.09 mol). The reaction was heated to 55° C. for 30 min.with a slow N₂ bubbling the reaction. The reaction was then concentratedin vacuo, basified with cold 2.0M NH₃ in IPA with CH₂Cl₂. The IPA/CH₂Cl₂was concentrated in vacuo and the salts were dissolved with minimalwater and extracted with CH₂Cl₂ (4×). The organic layers where combinedand washed with sat. NaHCO₃ (1×), dried over sodium sulfate, filteredand concentrated in vacuo. The crude product was purified via silica gelcolumn chromatography [eluting with 2:1 Hex:EtOAc] to yield the titlecompound as a light yellow solid; ¹H NMR (400 MHz, CDCl₃) δ 3.24-3.30(4H, m), 3.78-3.85 (1H, m), 4.89-4.94 (2H, m), 7.33 (1H, d, J=4.99 Hz),7.53 (1H, d, J=5.09 Hz), 7.82 (1H, s); MS (ES+): m/z 220.28/222.30(100/80) [MH⁺]; HPLC: t_(R)=2.87 min (OpenLynx, polar_(—)5 min).

3-Methylene-cyclobutanecarboxylic acid (3-chloropyrazin-2-ylmethyl)amide

C-(3-Chloropyrazin-2-yl)-methylamine bis-HCl (1.0 g, 4.62 mmol),N-ethyl-N′-(3-dimethylaminopropyl)carbodiimide (EDC) (1.31 g, 6.47 mmol,1.4 eq.), 4-dimethylamino pyridine (DMAP) (0.141 g, 1.15 mmol, 0.25eq.), and diisopropylethylamine (DIPEA) (2.42 mL, 1.79 g, 13.9 mmol, 3.0eq.) were dissolved in anhydrous CH₂Cl₂ (25 mL). To this solution, asolution of 3-methylenecyclobutanecarboxylic acid (0.622 g, 5.54 mmol,1.2 eq.) in anhydrous CH₂Cl₂ (25 mL) was added under N₂ and the reactionwas allowed to stir overnight at rt. Reaction mixture was concentratedin vacuo and the resulting residue was dissolved in EtOAc, washed withwater (2×), NaHCO₃ (1×), water (1×), and brine (1×), dried over Na₂SO₄,filtered, and concentrated in vacuo, giving crude title compound, as abrown oil. The crude material was purified by chromatography on silicagel [Jones Flashmaster, 20 g/70 mL cartridge, eluting with EtOAc:Hex10%→20%→40%→70%], affording the title compound as a pale yellow solid.Additionally, the title compound could be prepared by the followingroute: 1,1′-Carbonyldiimidazole (CDI) (0.824 g, 5.08 mmol, 1.1 eq.) and3-methylenecyclobutanecarboxylic acid (0.570 g, 5.08 mmol, 1.1 eq.) weredissolved in anhydrous THF (12 mL) and allowed to stir at 60° C. for 2h. A solution of C-(3-chloropyrazin-2-yl)-methylamine bis-HCl (1.0 g,4.62 mmol) and diisopropylethylamine (DIPEA) (2.42 mL, 1.79 g, 13.9mmol, 3.0 eq.) in anhydrous CH₂Cl₂ (13 mL) was added to the acid mixtureand the reaction was allowed to stir at 60° C., under N₂, overnight. Thereaction mixture was concentrated in vacuo and the resulting residue wasdissolved in EtOAc, washed with NaHCO₃ (2×) and brine (1×), dried overNa₂SO₄, filtered, and concentrated in vacuo, giving crude titlecompound, as a brown oil. The crude material was purified bychromatography on silica gel [Jones Flashmaster, 20 g/70 mL cartridge,eluting with EtOAc:Hex 10%→20%→40%→70%], affording the title compound asa pale yellow solid; ¹H NMR (CDCl₃, 400 MHz) δ 2.86-2.96 (m, 2H),3.03-3.19 (m, 3H), 4.72 (dd, J=4.4, 0.8 Hz, 2H), 4.79-4.84 (m, 2H), 6.78(s, —NH), 8.32-8.34 (m, 1H), 8.46 (d, J=2.8 Hz, 1H); MS (ES+): m/z238.19 (90) [MH⁺]; HPLC: t_(R)=2.67 min (OpenLynx, polar_(—)7 min).

3-(8-Amino-1-iodoimidazo[1,5-a]pyrazin-3-yl)cyclobutanol

In a Parr pressure reactor3-(8-chloro-1-iodo-imidazo[1,5-a]pyrazin-3-yl)-cyclobutanol (4.159 g,0.0119 mol) was dissolved with 2.0M ammonia in isopropyl alcohol (40mL). The mixture was cooled to −20° C. and saturated with ammonia. Thereaction was heated at 110° C. for 63 h at which point it was cooled andconcentrated in vacuo. The crude product was purified using HPFC Jones25 g silica gel column eluting with 5-8% MeOH: CH₂Cl₂ to yield the titlecompounds; MS (ES+): m/z 330.88 (100) [MH⁺], 331.89 (10) [MH⁺⁺]; HPLC:t_(R)=0.48 min (OpenLynx, polar_(—)5 min); ¹H NMR (CDCl₃, 400 MHz) δ2.55-2.76 (m, 2H) 3.06-3.22 (m, 2H) 3.32-3.50 (m, 1H) 4.51-4.69 (m, 1H)6.15 (br. s., 2H) 7.24 (d, J=5.05 Hz, 1H) 7.39 (d, J=5.05 Hz, 1H).

3-(8-Chloro-1-iodoimidazo[1,5-a]pyrazin-3-yl)cyclobutanol

3-(8-Chloro-1-iodo-imidazo[1,5-a]pyrazin-3-yl)-cyclobutanone (5.0 g, 14mmol) was dissolved in a 1:1 mixture of methanol (35.0 mL) and CH₂Cl₂(35.0 mL). To the solution mixture sodium tetrahydroborate (560 mg, 14.0mmol) was added slowly, gas evolution was observed. After 4.5 h at rtunder nitrogen, the reaction was concentrated in vacuo. The crude mixwas dissolved in EtOAc and washed with water. The organic layer wasdried over sodium sulfate, filtered and concentrated in vacuo. The crudeproduct was purified using HPFC Jones 50 gram silica gel column elutingwith 50% EtOAc: Hex to 100% EtOAc, to yield the title compound as alight yellow solid; MS (ES+): m/z 349.81 (100) [MH⁺], 351.50 (30)[MH⁺⁺⁺]; HPLC: t_(R)=2.49 min (OpenLynx, polar_(—)5 min); ¹H NMR (CDCl₃,400 MHz) δ 2.41-2.54 (m, 2H) 2.78-3.05 (m, 1H) 3.12-3.32 (m, 1H)4.08-4.75 (m, 1H) 5.30 (s, 1H) 7.31 (d, J=5.05 Hz, 1H) 7.57 (d, J=4.80Hz, 1H)

1-{4-[3-(8-Amino-1-iodoimidazo[1,5-a]pyrazin-3-yl)cyclobutyl]piperazin-1-yl}ethanone

1-{4-[3-(8-Chloro-1-iodoimidazo[1,5-a]pyrazin-3-yl)cyclobutyl]piperazin-1-yl}ethanone(13.2 g, 0.029 mol) was dissolved in isopropyl alcohol (100 mL) into aParr pressure reactor. The vessel was cooled to −78° C. and saturatedwith ammonia gas and sealed. The reaction was heated for 19 h at 110°C., at which point the reaction was cooled and the solvent concentratedin vacuo. The crude product was purified via silica gel chromatographyeluting with 5-10% MeOH (7M NH₃): CH₂Cl₂ to yield the title compounds asan off white solid; MS (ES+): m/z 440.89 (100) [MH⁺], 441.89 (20)[MH⁺⁺]; HPLC: t_(R)=0.46 min (OpenLynx, polar_(—)5 min); ¹H NMR (CDCl₃,400 MHz) δ 2.09 (s, 3H) 2.28-2.48 (m, 6H) 2.54-2.71 (m, 2H) 2.80-2.99(m, 1H) 3.27-3.43 (m, 1H) 3.43-3.54 (m, 2H) 3.56-3.70 (m, 2H) 7.02 (d,J=5.05 Hz, 1H) 7.16 (d, J=5.05 Hz, 2H).

1-{4-[3-(8-Chloro-1-iodoimidazo[1,5-a]pyrazin-3-yl)cyclobutyl]piperazin-1-yl}ethanone

Into a RBF 3-(8-chloro-1-iodoimidazo[1,5-a]pyrazin-3-yl)cyclobutanone(1.00 g, 0.0029 mol) and sodium triacetoxyborohydride (1.30 g, 0.006mol) were dissolved in 1,2-dichloroethane (65.0 mL) and a solution of1-acetylpiperazine (0.39 g, 0.003 mol) in 1,2-dichloroethane was addedto the reaction. The reaction mixture was stirred at rt for 2 h. Thecrude product was concentrated in vacuo and the dissolved in CH₂Cl₂(25.0 mL) and washed with saturated NaHCO₃ solution (1×40 mL). Theproduct was dried with sodium sulfate and concentrated in vacuo to yielda light yellow solid; MS (ES+): m/z 459.84 (100) [MH⁺], 461.80 (40)[MH⁺⁺⁺]; HPLC: t_(R)=1.81 min (OpenLynx, polar5 min); ¹H NMR (CDCl₃, 400MHz) δ 2.04-2.15 (m, 3H) 2.26-2.50 (m, 6H) 2.55-2.72 (m, 2H) 2.83-2.99(m, 1H) 3.29-3.52 (m, 3H) 3.56-3.67 (m, 2H) 7.29 (d, 1H) 7.58 (d, 1H).

(1-Iodo-3-[3-(4-methyl-piperazin-1-yl)-cyclobutyl]-imidazo[1,5-a]pyrazin-8-ylamine)

A solution of 2N ammonia in isopropyl alcohol (350 mL) and THF (30 mL,0.4 mol) was added to8-chloro-1-iodo-3-[3-(4-methyl-piperazin-1-yl)-cyclobutyl]-imidazo[1,5-a]pyrazine(19.91 g, 0.04612 mol) in a Parr bomb and cooled to −78° C. Ammonia wasbubbled into the solution for 8-10 min. The bomb was sealed, stirred andheated to at 110° C. over 3d. The solvent was then evaporated in vacuoand purified by flash silica gel chromatography (wetted with CHCl₃,dried loaded with silica, and eluted with 8% (7N NH₃) MeOH in CHCl₃),which afforded the title compound; ¹H NMR (CDCl₃, 400 MHz) δ 7.31 (1H,d, J=5.01), 7.16 (1H, d, J=6.25), 5.83 (2H, s), 3.49 (1H, m), 3.06 (1H,m), 2.76 (4H, m), 2.64 (8H, m), 2.46 (3H, s); MS (ES+): m/z412.89/413.91 (50/10) [MH⁺]; HPLC: t_(R)=0.31 min. (OpenLynx, polar_(—)5min.).

(8-Chloro-1-iodo-3-[3-(4-methylpiperazin-1-yl)cyclobutyl]imidazo[1,5-a]pyrazine)

1-Methyl piperazine (5.75 mL, 0.0514 mol) in 1,2-dichloroethane (1096.7mL, 13.892 mol) was added to3-(8-chloro-1-iodoimidazo[1,5-a]pyrazin-3-yl)cyclobutanone (17.00 g,0.04892 mol) and sodium triacetoxyborohydride (21.8 g, 0.0978 mol). Thereaction stirred at rt for 3 h. The reaction was concentrated, dissolvedin CH₂Cl₂, and then washed with saturated NaHCO₃ solution and brine. Theproduct was dried over sodium sulfate, filtered, and concentrated invacuo. The product was flushed through a quick silica gel plug (wettedwith 100% CHCl₃, eluted with 8% (7N NH₃) MeOH in CHCl₃), to afford thetitle compound; ¹H NMR (CDCl₃, 400 MHz) δ 7.63 (1H, d), 7.30 (1H, d),3.42 (1H, m), 2.94 (1H, m), 2.65 (4H, m), 2.44 (8H, m), 2.32 (3H, s); MS(ES+): m/z 431.85/433.87 (100/45) [MH⁺]; HPLC: t_(R)=1.82 min.(OpenLynx, polar_(—)5 min.).

3-(8-Chloroimidazo[1,5-a]pyrazin-3-yl)-1-methylcyclobutanol

3-(8-Chloroimidazo[1,5-a]pyrazin-3-yl)cyclobutanone (1.95 g, 8.80 mmol)in anhydrous THF (77.78 mL) at −78° C. under an atmosphere of nitrogenwas treated slowly with a 3.0 M solution of methylmagnesium chloride inTHF (5.9 mL). The solution stirred for 3 hr at −78° C. then quenchedwith 40 mL of semi-saturated aqueous NH₄Cl (NH₄Cl dilution in 1:1mixture with water) at −78° C. and allowed to warm up to rt. The mixturewas then extracted with EtOAc (3×40 mL) and the combined extracts washedwith brine (30 mL), dried over magnesium sulfate, filtered andconcentrated in vacuo. The crude solid was purified by chromatographyover silica gel eluting with 1:1 EtOAc/DCM to 4% MeOH in (1:1) EtOAc/DCMto afford desired product. ¹H-NMR (400 MHz, CDCl₃) δ ppm 1.54 (s, 3H),2.74-2.60 (m, 4H), 3.75-3.39 (m, 1H), 7.35 (d, J=5.04 Hz, 1H), 7.71 (d,J=5.00 Hz, 1H) and 7.86 (s, 1H). MS (ES+): m/z 238.15 and 240.17 [MH⁺].

3-(8-Chloro-1-iodoimidazo[1,5-a]pyrazin-3-yl)-1-methylcyclobutanol

3-(8-Chloroimidazo[1,5-a]pyrazin-3-yl)-1-methylcyclobutanol (2.20 g,9.26 mmol) and NIS (2.71 g, 12.0 mmol) were dissolved in DMF (36.6 mL,0.472 mol) and stirred at 60° C. for 4 h. The mixture was thenconcentrated in vacuo and the residue reconstituted in EtOAc (100 mL).This solution was washed with sodium bicarbonate (2×20 mL) and thesewashes back-extracted with EtOAc (2×20 mL). The organic layers werecombined, dried with sodium sulfate, filtered and concentrated in vacuo.The crude solid was purified by chromatography over silica gel elutingwith 1:1 EtOAc:hexanes to afford desired product. ¹H-NMR (400 MHz,CDCl₃) δ ppm 1.53 (s, 3H), 2.72-2.59 (m, 4H), 3.37-3.29 (m, 1H), 7.32(d, J=4.91 Hz, 1H) and 7.60 (d, J=4.96 Hz, 1H). MS (ES+): m/z 363.95 and365.91 [MH⁺].

3-(8-Amino-1-iodoimidazo[1,5-a]pyrazin-3-yl)-1-methylcyclobutanol

A solution of 2M ammonia in isopropanol (80 mL) and THF (5 mL) was addedto 3-(8-chloro-1-iodoimidazo[1,5-a]pyrazin-3-yl)-1-methylcyclobutanol(2.77 g, 7.62 mmol) in a Parr pressure reactor. The mixture was cooledto at −78° C. then ammonia gas was bubbled into the solution for 4-6min. The reactor was sealed then heated at 110° C. for 15 h. The solventwas then removed in vacuo and the residue purified by chromatographyover silica gel eluting with 7% MeOH in DCM to afford desired product.¹H NMR (400 MHz, DMSO-d6) δ ppm 1.44 (s, 3H), 2.32-2.51 (m, 4H),3.33-3.52 (m, 1H), 6.61 (br.s., 2H), 7.03 (d, J=5.05 Hz, 1H) and 7.62(d, J=5.05 Hz, 1H).

(3-(8-Chloro-1-iodoimidazo[1,5-a]pyrazin-3-yl)cyclobutanone)

A solution of3-(8-chloro-1-iodoimidazo[1,5-a]pyrazin-3-yl)-1-hydroxymethylcyclobutanol(4.08 g, 0.01 μmol) in THF (120 mL) and water (40 mL) was charged withsodium periodate (2.8 g, 0.013 mol) at 0° C. The reaction warmed to rtand stirred for 5 h. The reaction mixture was diluted with ethyl acetateand then washed with brine. The organic phase was dried over Na₂SO₄,filtered, and concentrated in vacuo to afford the title compound as ayellow solid; ¹H NMR (CDCl₃, 400 MHz) δ 7.56 (1H, d, J=4.94), 7.32 (1H,d, J=4.98), 3.64 (5H, m); MS (ES+): m/z 347.82 and 349.85 [MH⁺]; HPLC:t_(R)=2.89 min. (OpenLynx, polar_(—)5 min.).

3-(8-Chloro-1-iodoimidazo[1,5-a]pyrazin-3-yl)-1-hydroxymethylcyclobutanol

Under inert atmosphere N-iodosuccinimide (3.6 g, 0.016 mol) and3-(8-chloroimidazo[1,5-a]pyrazin-3-yl)-1-hydroxymethylcyclobutanol (3.16g, 0.012 mol) were dissolved in N,N-dimethylformamide (30 mL) and heatedat 60° C. for 3.0 h. The reaction mixture was then concentrated in vacuoto a dark oil and purified by HPFC Jones 20 g silica gel column, elutingwith 5% MeOH: CH₂Cl₂ to yield a light brown fluffy solid which wastriturated with diethyl ether and hexanes to afford the title compound;MS (ES+): m/z 379.85 and 381.80 [MH⁺]; HPLC: t_(R)=2.30 min (OpenLynx,polar_(—)5 min).

3-(8-Chloroimidazo[1,5-a]pyrazin-3-yl)-1-hydroxymethylcyclobutanol

To a THF solution (170 mL) of8-chloro-3-(3-methylenecyclobutyl)imidazo[1,5-a]pyrazine (3.1 g, 14mmol), water (18 mL), 50% N-methylmorpholine-N-oxide in water (3.2 mL)and potassium osmate, dehydrate (200 mg, 0.70 mmol) were added and thereaction was allowed to stir at rt for 4 h.

Sodium sulfite (8.0 g, 70.0 mmol) was added to the reaction mixture andallowed to stir for 30 min at which point the reaction was concentratedin vacuo. The crude product was extracted from the aqueous with EtOAc.The organics were washed with brine and the combined aqueous washes wereback extracted with EtOAc (5×50 mL). The combined organics were driedover sodium sulfate, filtered, and concentrated in vacuo to yield thetitle compounds as a sticky tan/off-white solid; MS (ES+): m/z 254.17(100) [MH⁺], 256.19 (50) [MH⁺⁺⁺]; HPLC: t_(R)=1.95 min (OpenLynx,polar_(—)5 min).

3-Methylene-cyclobutanecarboxylic acid

To a solution of 3-methylenecyclobutanecarbonitrile (100.0 g, 1.042 mol)in ethanol (1.00 L) and water (1.00 L) was added potassium hydroxide(230.0 g, 4.2 mol). The resulting mixture was heated at reflux for 7 hrthen the EtOH was removed in vacuo and the solution was cooled to 0° C.and acidified with (300.0 mL) of conc. HCl to pH=1. The mixture wasextracted with diethyl ether (4×1 L) and the combined organic phaseswere dried over sodium sulfate, filtered and concentrated in vacuo toyield desired product. ¹H NMR (400 MHz, CDCl₃) δ ppm 2.64-3.44 (m, 5H),4.60-4.98 (m, 2H) and 10.64 (br. s., 1H).

Ethyl 3-methylenecyclobutanecarboxylate

Iodoethane (7.5 mL, 93.0 mol) was added at rt to a mixture of3-methylenecyclobutanecarboxylic acid (10.0 g, 80.0 mmol) and cesiumcarbonate (56.0 g, 170.0 mmol) in anhydrous N,N-dimethylformamide(500.00 mL) under an atmosphere of nitrogen. The reaction was stirredfor 16 hr then partitioned between diethyl ether (1 L) and brine (1 L).The aqueous layer was extracted with diethyl ether (3×500 mL) and thecombined organic phases washed with water (2×1 L), dried over sodiumsulfate, filtered and concentrated in vacuo to yield desired product ¹HNMR (400 MHz, CDCl₃) δ ppm 1.26 (t, 3H), 2.71-3.27 (m, 5H), 4.15 (q,J=7.07 Hz, 2H) and 4.53-4.96 (m, 2H).

N-[(3-chloropyrazin-2-yl)methyl]-3-methylenecyclobutanecarboxamide

1,1′-Carbonyldiimidazole (CDI) (8.24 g, 50.81 mmol) and3-methylenecyclobutanecarboxylic acid (5.70 g, 50.81 mmol) weredissolved in anhydrous THF (100 mL) and allowed to stir at 60° C. for 4h. A solution of C-(3-Chloropyrazin-2-yl)methylamine bis-hydrochloride(10.0 g, 46.19 mmol) and diisopropylethylamine (DIPEA) (32.30 mL, 184.76mmol) in anhydrous CH₂Cl₂ (150 mL) was added to the mixture and thereaction was allowed to stir at rt for 24 h. The mixture wasconcentrated in vacuo, the residue dissolved in EtOAc and the resultingsolution washed with saturated NaHCO₃ (aq.) water H₂O and Brine. Thecombined organic layers were dried over sodium sulfate, filtered andconcentrated in vacuo to afford crude product, which was purified bychromatography over silica gel eluting with 50-70% EtOAc/hexane to yielddesired product. ¹H NMR (400 MHz, CDCl₃) δ ppm 2.92-2.94 (2H, m),3.05-3.14 (2H, m), 4.60 (2H, d, J=4.24 Hz), 4.80-4.84 (2H, m), 6.75 (1H,brs), 8.33 (1H, d, J=4.22 Hz) and 8.45 (1H, d, J=2.54 Hz). MS (ES+): m/z238 and 240 [MH+].

8-Chloro-3-(3-methylenecyclobutyl)imidazo[1,5-a]pyrazine

N-[(3-Chloropyrazin-2-yl)methyl]-3-methylenecyclobutanecarboxamide (52.1g, 219.2 mmol) in anhydrous MeCN (1.0 L) was treated with DMF (1.0 mL)and POCl₃ (100 mL, 1.09 mol) and the mixture was stirred at 55° C. for30 min. under a gentle stream of N₂. The reaction was then concentratedin vacuo and the residue reconstituted in CH₂Cl₂ and treated with cold2.0 M NH₃ in IPA. This mixture was concentrated in vacuo, water added todissolve the salts, and then extracted with CH₂Cl₂ (4×60 mL). Theorganic layers where combined and washed with sat. NaHCO₃ (1×70 mL)dried over sodium sulfate, filtered and concentrated in vacuo. The crudematerial was purified by chromatography over silica gel eluting with 2:1hexane:EtOAc to yield desired product. ¹H NMR (400 MHz, CDCl₃) δ ppm3.24-3.30 (4H, m), 3.78-3.85 (1H, m), 4.89-4.94 (2H, m), 7.33 (1H, d,J=4.99 Hz), 7.53 (1H, d, J=5.09 Hz) and 7.82 (1H, s). MS (ES+): m/z220.28 and 222.30 [MH+].

C-(3-Chloropyrazin-2-yl)methylamine bis-hydrochloride

A solution of 2-(3-chloropyrazin-2-ylmethyl)-isoindole-1,3-dione (10.0g, 36.5 mmol) in anhydrous CH₂Cl₂ (200 mL) was charged with hydrazine(2.87 mL, 2.93 g, 91.3 mmol, 2.5 eq.) at rt, under N₂ atmosphere. After2.5 h, MeOH (300 mL) was added and the reaction was heated until thesolution was homogenous. The reaction mixture was allowed to stir for 19h. The white ppt that had formed (2,3-dihydrophthalazine-1,4-dionebyproduct), was filtered off and washed several times with ether. Theclear filtrate was concentrated in vacuo and the concentrate wasdissolved in EtOAc and filtered again to remove white ppt. All solventwas removed, giving a yellow oil, which was dissolved into EtOAc andether and charged with HCl (g). The title compound, a pale yellow solid,instantly precipitated. The title compound was dried in a 40° C. ovenfor 72 h, affording the title compound, as a dark yellow solid; ¹H NMR(400 MHz, CD₃OD) δ 4.55 (2H, s), 8.27 (1H, d, J=2.52 Hz), 8.54 (1H, d,J=2.56 Hz); MS (ES+): m/z 143.96/145.96 (100/60) [MH⁺]; HPLC: t_(R)=0.41min (OpenLynx, polar_(—)7 min).

1-{[(3-Oxocyclobutyl)carbonyl]oxy}pyrrolidine-2,5-dione

Into a 5 L reactor equipped with a nitrogen flow and an overhead stirrerwas added N-hydroxysuccinimide (250.0 g, 2.172 mol) and3-oxo-cyclobutanecarboxylic acid (248 g, 2.17 mol). Ethyl acetate (3.4L) was added and the reaction was cooled to 16° C. A solution of 25% DCCin EtOAc (2.17 mol) was added slowly via an addition funnel to thereaction mixture over 7 minutes then the mixture was then heated at 45°C. After 2 h, the mixture was filtered and the filtrate was washed oncewith EtOAc (1 L×1) and evaporated to dryness in vacuo to afford thedesired product. ¹H NMR (400 MHz, DMSO-d6) δ 2.83 (bs, 4H), 3.30-3.39(m, 2H), 3.52-3.60 (m, 2H) and 3.67-3.73 (m, 1H).

3-(8-Chloroimidazo[1,5-a]pyrazin-3-yl)cyclobutanone

Into a round bottom 1-neck flask (5 L), 3-oxo-cyclobutanecarboxylic acid2,5-dioxo-pyrrolidin-1-yl ester (217.2 g, 0.937 mol),C-(3-chloro-pyrazin-2-yl)-methylamine hydrochloride salt (153.3 g, 0.852mol), and THF (760 mL) were added. A solution of 10% NaHCO3 (1.07 kg)was then added and after 20 min, the layers were allowed to separate andthe aqueous layer was removed. The aqueous layer was back extracted withEtOAc (1×700 mL, 1×300 mL). The combined organics were washed with brine(350 mL), dried over MgSO₄, filtered, and concentrated in vacuo toprovide the title compound. This solid was resuspended in ethyl acetate(915 mL) and DMF (132 mL) and the solution was put under an atmosphereof nitrogen and cooled to 10.5° C. Phosphorus oxychloride (159 mL, 1.70mol) was then added over 15 minutes and the reaction was allowed to stirfor 45 min. The reaction solution was then poured slowly into a 22%aqueous Na₂CO₃ solution at 10° C. Water (1 L) was added and the layerswere allowed to separate. The organic layer was removed and the aqueouswas back extracted with EtOAc (1×1 L, 1×0.5 L). The combined organicphases were dried over MgSO₄, filtered, and concentrated in vacuo untilabout 0.5 L of solvent remained. Heptane was added and the slurry wasconcentrated in vacuo until most of the EtOAc was removed. The resultantslurry was filtered to give desired product. ¹H NMR (400 MHz, CDCl₃) δ3.59-3.68 (m, 2H), 3.72-3.79 (m, 2H), 3.86-3.94 (m, 1H), 7.40 (d, 1H,J=5.2 Hz), 7.60 (d, 1H, J=5.2 Hz) and 7.85 (s, 1H).

3-(1-Bromo-8-chloroimidazo[1,5-a]pyrazin-3-yl)cyclobutanone

3-(8-Chloroimidazo[1,5-a]pyrazin-3-yl)cyclobutanone (47.7 g, 215 mmol)was dissolved in DMF (200 mL) under an atmosphere of nitrogen and cooledto −4° C. N-Bromosuccinimide (40.3 g, 226 mmol) was dissolved in DMF(140 mL) and slowly added to the reaction mixture. After 5 min, water(400 mL) was added and the resulting solid isolated by filtration andwashed with solid with water to give the title compound. ¹H NMR(DMSO-d6, 400 MHz): δ 3.45-3.53 (m, 2H), 3.58-3.67 (m, 2H), 4.08-4.16(m, 1H), 7.45 (d, 1H, J=5.2 Hz) and 8.30 (d, 1H, J=4.8 Hz).

3-(1-Bromo-8-chloroimidazo[1,5-a]pyrazin-3-yl)-1-methylcyclobutanol

3-(1-Bromo-8-chloroimidazo[1,5-a]pyrazin-3-yl)cyclobutanone (51.988 g,0.17 mol) in anhydrous THF (550 g, 620 mL) under nitrogen at −78° C. wastreated with a 3.0 M solution of methyl magnesium chloride in THF (130mL, 0.38 mol) over min. The mixture was stirred at −78° C. for 30 minand then the cooling bath was removed and the mixture quenched with 14%NH₄Cl (132 g). EtOAc was added to the aqueous phase and the pH wasadjusted to −5 with 20% HCl and the layers separated. The combinedorganic phases were concentrated in vacuo to a slurry and 0.5 L oftoluene was added and the mixture concentrated in vacuo until the EtOAcwas removed. The slurry was heated at reflux until homogeneous thenallowed to cool to provide desired product, which was isolated byfiltration and dried in vacuo. ¹H NMR (DMSO-d₆, 400 MHz): δ 1.37 (s,3H), 2.35-2.49 (m, 4H), 3.52 (dddd, 1H, J=9.6, 9.6, 9.6, 9.6 Hz), 5.18(bs, 1H), 7.37 (d, 1H, J=5.2 Hz) and 8.26 (d, 1H, J=5.2 Hz).

3-(8-Amino-1-bromoimidazo[1,5-a]pyrazin-3-yl)-1-methylcyclobutanol

A 35% ammonia solution (132 ml, 2.9 moles) was added to a suspension of3-(1-bromo-8-chloroimidazo[1,5-a]pyrazin-3-yl)-1-methylcyclobutanol(22.0 g, 0.06463 mol) in 2-butanol (81 ml). The mixture was heated at90° C. in a pressure vessel for 15 hr then concentrated to ˜130 ml,cooled to room temperature and the solid collected by filtration. Thismaterial was washed with water (3×22 mL) and dried at 40° C. undervacuum. To afford the desired product. ¹H NMR (DMSO-d₆, 400 MHz): δ 7.5(d, 1H), 7.0 (d, 1H), 6.6 (bs, 2H), 5.1 (s, 1H), 3.4 (pentet, 1H),2.3-2.4 (m, 4H) and 1.4 (s, 3H).

7-Cyclobutyl-5-iodoimidazo[5,1-f][1,2,4]triazin-4-ylamine

To a solution of 1,2,4-triazole (1.28 g, 18.59 mmol) in anhydrouspyridine (10 mL) was added phosphorus oxychloride (POCl₃) (0.578 mL,6.20 mmol) and stirred at rt for 15 min. This mixture was dropwisecharged (3.5 min) with a solution of 7-cyclobutyl-5-iodo-3Himidazo[5,1f][1,2,4]triazin-4-one (0.653 mg, 2.07 mmol) in anhydrouspyridine (14 mL) and stirred for 1.5 h. The reaction mixture was cooledto 0° C. quenched with 2M NH₃ in isopropanol (IPA) until basic thenallowed to reach rt and stirred for an additional 2 h. The reactionmixture was filtered through a fritted Buchner funnel and washed withDCM. The filtrate was concentrated in vacuo and purified bychromatography on silica gel [eluting with 30% EtOAc in DCM]resulting inthe title compound as an off-white solid; ¹H NMR (CDCl₃, 400 MHz) δ1.93-2.04 (m, 1H), 2.05-2.18 (m, 1H), 2.35-2.45 (m, 2H), 2.49-2.62 (m,2H), 4.00-4.12 (m, 1H), 7.82 (s, 1H); MS (ES+): m/z 316.08 (100) [MH⁺],HPLC: t_(R)=2.59 min (MicromassZQ, polar_(—)5 min).

7-Cyclobutyl-5-iodo-3H-imidazo[5,1-f][1,2,4]triazin-4-one

A solution of 7-cyclobutyl-3H-imidazo[5,1-f][1,2,4]triazin-4-one (789mg, 4.15 mmol) and N-iodosuccinimide (NIS, 933 mg, 4.15 mmol) inanhydrous DMF (40 mL) was stirred overnight at rt. An additional 4 eq.of NIS was added and reaction was heated to 55° C. for 6 h. The reactionmixture was concentrated in vacuo and partitioned between DCM and H₂Oand separated. The aqueous layer was washed with DCM (3×) and thecombined organic fractions were washed with 1M sodium thiosulfate(Na₂S₂O₃) (1×), brine (1×), dried over sodium sulfate (Na₂SO₄),filtered, and concentrated in vacuo. The solid was triturated with 20%EtOAc in DCM and filtered through a fritted Buchner funnel resulting inthe title compound as an off-white solid; ¹H NMR (DMSO-d₆, 400 MHz) δ1.84-1.96 (m, 1H), 1.98-2.13 (m, 1H), 2.25-2.43 (m, 4H), 3.84-3.96 (m,1H), 7.87 (s, 1H); MS (ES+): m/z 317.02 (100) [MH⁺], HPLC: t_(R)=2.62min (MicromassZQ, polar_(—)5 min).

7-Cyclobutyl-3H-imidazo[5,1-f][1,2,4]triazin-4-one

A crude solution of cyclobutanecarboxylic acid(5-oxo-4,5-dihydro-[1,2,4]triazin-6-ylmethyl)amide (1.33 g, 6.39 mmol)in phosphorus oxychloride (POCl₃) (10 mL) was heated to 55° C. Thereaction was heated for 2 h then concentrated in vacuo and the crude oilwas cooled to 0° C. in an ice-bath and quenched with 2M NH₃ inispropanol (IPA) until slightly basic. This crude reaction mixture wasconcentrated in vacuo and was partitioned between DCM and H₂O andseparated. The aqueous layer was extracted with DCM (3×) and thecombined organic fractions were dried over sodium sulfate (Na₂SO₄),filtered and concentrated in vacuo. The crude material was purified bychromatography on silica gel [eluting with 5% MeOH in DCM], resulting inthe title compound as an off-white solid; ¹H NMR (DMSO-d₆, 400 MHz) δ1.86-1.96 (m, 1H), 2.00-2.13 (m, 1H); 2.26-2.46 (m, 4H); 3.87-4.00 (m,1H); 7.71 (s, 1H); 7.87 (d, J=3.6 Hz, 1H); 11.7 (brs, 1H); MS (ES+): m/z191.27 (100) [MH⁺], HPLC: t_(R)=2.06 min (MicromassZQ, polar_(—)5 min).

Cyclobutanecarboxylic acid(5-oxo-4,5-dihydro-[1,2,4]triazin-6-ylmethyl)amide

To a solution of 6-aminomethyl-4H-[1,2,4]triazin-5-one (500 mg, 3.96mmol) and N,N-diisopropylethylamine (DIEA) (0.829 mL, 4.76 mmol) inanhydrous N,N-dimethylforamide (DMF) (20 mL) and anhydrous pyridine (2mL) was dropwise charged with cyclobutanecarbonyl chloride (0.451 mL,3.96 mmol) at 0° C. then warmed to rt and stirred for an additional 1.5h. The reaction mixture was quenched with H₂O (2 mL) and concentrated invacuo and was purified by chromatography on silica gel [eluting with 5%MeOH in DCM (200 mL)→10% MeOH in DCM (800 mL)], affording the titlecompound; ¹H NMR (DMSO-d₆, 400 MHz) δ 1.7-1.82 (m, 1H), 1.70-1.92 (m,1H); 1.97-2.07 (m, 2H); 2.07-2.19 (m, 2H); 3.55-3.67 (m, 1H); 4.19 (d,2H); 7.97 (brt, J=5.6 Hz, 1H); 8.67 (s, 1H); MS (ES+): m/z 209.25 (100)[MH⁺], HPLC: t_(R)=1.56 min (MicromassZQ, polar_(—)5 min).

6-Aminomethyl-4H-[1,2,4]triazin-5-one

A slurry of2-(5-oxo-4,5-dihydro-[1,2,4]triazin-6-ylmethyl)isoindole-1,3-dione (4 g,15.6 mmol) in DCM/EtOH (1:1) (150 mL) was charged with anhydroushydrazine (1.23 mL, 39.0 mmol) and stirred at rt for 18 h. The reactionmixture was concentrated in vacuo and the off-white solid was trituratedwith warm CHCl₃ and filtered through a fritted funnel. The solid wasthen triturated with hot boiling methanol (MeOH) and filtered through afritted funnel resulting in an off-white solid. The material wastriturated a second time as before and dried overnight resulting in thetitle compound as a white solid, which was taken on to the next stepwithout further purification; ¹H NMR (DMSO-d₆, 400 MHz) δ 3.88 (s, 2H),8.31 (2, 1H); MS (ES+): m/z 127.07 (100) [MH⁺], HPLC: t_(R)=0.34 min(MicromassZQ, polar_(—)5 min).

2-(5-Oxo-4,5-dihydro-[1,2,4]triazin-6-ylmethyl)isoindole-1,3-dione

A slurry of2-(5-oxo-3-thioxo-2,3,4,5-tetrahydro-[1,2,4]triazin-6-ylmethyl)isoindole-1,3-dione(1.0 g, 3.47 mmol) in EtOH (40 mL) was charged with excess Raney Ni (3spatula) and heated to reflux for 2 h. The reaction mixture was filteredhot through a small pad of celite and washed with a hot mixture ofEtOH/THF (1:1) (100 mL) and the filtrate was concentrated in vacuoresulting in the title compound as an off-white solid; ¹H NMR (DMSO-d₆,400 MHz) δ 4.75 (s, 2H), 7.84-7.98 (m, 4H), 8.66 (s, 1H); MS (ES+): m/z257.22 (100) [MH⁺].

2-(5-Oxo-3-thioxo-2,3,4,5-tetrahydro-[1,2,4]triazin-6-ylmethyl)indan-1,3-dione

A slurry of 3-(1,3-dioxo-1,3-dihydroisoindol-2-yl)-2-oxo-propionic acidethyl ester (20 g, 76.6 mmol) in anhydrous EtOH (300 mL) was chargedwith thiosemicarbazide (6.98 g, 76.6 mmol) in one portion and heated to80° C. for 2 h. The reaction mixture was charged withN,N-diisopropylethylamine (DIEA) (26.7 mL, 76.56 mmol) and heated to 40°C. for 6 h then stirred at rt for an additional 10 h. The reactionmixture was concentrated in vacuo and solid was triturated with hotEtOH/EtOAc filtered and washed with EtOAc. The solid was dried overnightin a vacuum oven (40° C.) resulting in the title compound as anoff-white solid; ¹H NMR (DMSO-d₆, 400 MHz) δ 4.68 (s, 2H), 7.85-7.95 (m,4H); MS (ES+): m/z 289.2 (100) [MH⁺].

2-[(3-Methyl-5-oxo-4,5-dihydro-1,2,4-triazin-6-yl)methyl]-1H-isoindole-1,3(2H)-dione

A solution of ethyl3-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)-2-oxopropanoate [J. Org.Chem., (1985), 50 (1), 91](4.29 g, 16.4 mmol), acetamidrazonehydrochloride (1.80 g, 16.4 mmol) in anhydrous EtOH (85.8 mL) was heatedto 80° C. for 3 h then cooled to rt and stirred for an additional 16 h.The reaction mixture was filtered through a fritted funnel resulting in3.28 g, (73% yield) of the title compound as a white solid. ¹H NMR (400MHz, DMSO-d6) δ ppm 2.28 (s, 3H), 4.73 (s, 2H) and 7.74-8.12 (m, 4H); MS(ES+): m/z 271.08 [MH+].

6-(Aminomethyl)-3-methyl-1,2,4-triazin-5(4H)-one

A solution of2-[(3-methyl-5-oxo-4,5-dihydro-1,2,4-triazin-6-yl)methyl]-1H-isoindole-1,3(2H)-dione(2.00 g, 7.40 mmol) in DCM (10.0 mL) and EtOH (10.0 mL) was charged withhydrazine (0.58 mL, 18.5 mmol) and stirred at rt for 8 h, then heated to45° C. for an additional 16 h. The reaction was charged with anadditional 0.5 equiv of hydrazine (0.116 mL, 3.70 mmol) and heated to45° C. for 4 h. The reaction mixture was allowed to cool to rt thenfiltered through a fritted funnel and the cake was washed with 2portions of cold 1:1 EtOH/DCM (75 mL) and the filtrate was concentratedresulting in 622 mg of a pale yellow solid which was taken on to thenext step without further purification. ¹H NMR (400 MHz, DMSO-d₆) δ ppm2.21 (s, 3H), 3.72 (s, 2H); MS (ES+): m/z 141.06 [MH+].

trans-4-({[(Benzyloxy)carbonyl]amino}methyl)cyclohexanecarboxylic acid

trans-4-(Aminomethyl)cyclohexanecarboxylic acid (10.00 g, 0.06361 mol),in a 10% aq solution of NaOH (5.60 g in 55 mL) was cooled to 0° C. andtreated over 15 min with vigorous stirring, with benzyl chloroformate(11 mL, 0.076 mol).

After one hour the solution was acidified (1M HCl(aq)) and the resultingthe white precipitate collected by filtration, washed with water andhexane then dried in vacuo oven overnight to afford 17.23 g of the titlecompound. ¹H NMR (400 MHz, CDCl₃): δ 0.93-0.99 (m, 2H), 1.38-1.46 (m,2H), 1.82-1.85 (m, 2H), 2.03-2.06 (m, 2H), 2.25 (m, 1H), 3.06 (t, J=5.6Hz, 2H), 4.83 (m, 1H), 5.09 (s, 2H), 7.31-7.36 (m, 5H). MS (ES+): m/z292 [MH+].

Benzyl[(trans-4-{[(3-chloropyrazin-2-yl)methyl]carbamoyl}cyclohexyl)methyl]carbamate

To a solution of C-(3-chloropyrazin-2-yl)methylamine hydrochloride salt(0.100 g, 0.533 mmol) in DCM (1.35 mL) was addedN-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (0.16 g,0.83 mmol), N,N-diisopropylethylamine (0.14 mL, 0.83 mmol),1-hydroxybenzotriazole (0.075 g, 0.56 mmol) andtrans-4-({[(benzyloxy)carbonyl]amino}methyl)cyclohexanecarboxylic acid(0.21 g, 0.70 mmol). The reaction was stirred at rt overnight thendiluted with DCM, washed with sat. NaHCO₃ (aq) and brine, then driedover Na₂SO₄ and the solvent removed in vacuo. The residue thus isolatedwas chromatographed over silica gel eluting with EtOAc/hexane (1:1) toafford 0.173 g of the title compound. ¹H NMR (400 MHz, CDCl₃): δ1.00-1.03 (m, 2H), 1.45-1.51 (m, 2H), 1.83-1.89 (m, 2H), 1.99-2.03 (m,2H), 2.20 (m, 1H), 3.05-3.12 (m, 3H), 4.68 (d, J=4.4 Hz, 2H), 4.79 (br,1H), 5.10 (s, 2H), 6.79 (br, 1H), 7.31-7.37 (m, 5H), 8.33 (d, J=2.8 Hz,1H), 8.46 (d, J=2.8 Hz, 1H). MS (ES+): m/z 417.14 [MH+].

Benzyl{[trans-4-(8-chloroimidazo[1,5-a]pyrazin-3-yl)cyclohexyl]methyl}carbamate

To a suspension of benzyl[(trans-4-{[(3-chloropyrazin-2-yl)methyl]carbamoyl}cyclohexyl)methyl]carbamate(0.100 g, 0.220 mmol) in EtOAc (0.9 mL) and DMF (0.068 mL) at 0° C. wasadded slowly POCl₃ (0.082 mL, 0.88 mmol). After stirring at rt for anhour, the mixture was cooled to 0° C. and solid NaHCO₃ was added. Aftera further 10 min at 0° C. and 20 min at rt, the mixture was re-cooled to0° C. and water (20 mL) was added. The reaction mixture was extractedwith EtOAc (3×20 mL) and the extracts washed with water (2×30 mL) andbrine (30 mL) and then dried over Na₂SO₄ and concentrated in vacuo toafford 0.096 g of the title compound. ¹H NMR (400 MHz, CDCl₃): δ1.15-1.19 (m, 2H), 1.76-1.87 (m, 3H), 1.93-2.00 (m, 2H), 2.04-2.08 (m,2H), 3.07 (m, 1H), 3.15 (t, J=6.4 Hz, 2H), 4.84 (br, 1H), 5.09 (s, 2H),7.31-7.40 (m, 6H), 7.61 (d, J=4.8 Hz, 1H), 7.79 (s, 1H). MS (ES+): m/z399.26 [MH+].

Benzyl{[trans-4-(8-chloro-1-iodoimidazo[1,5-a]pyrazin-3-yl)cyclohexyl]methyl}carbamate

To a solution of benzyl{[trans-4-(8-chloroimidazo[1,5-a]pyrazin-3-yl)cyclohexyl]methyl}carbamate(1.49 g, 0.00374 mol) in DMF (0.6 mL) was added NIS (1.0 g, 0.0045 mol).The reaction mixture was stirred at 55° C. overnight then diluted withEtOAc (20 mL), washed with water (2×40 mL) and brine (20 mL), then driedover Na₂SO₄ and concentrated in vacuo. The crude mixture thus isolatedwas chromatographed over silica gel eluting with hexane→hexane:EtOAc 1:1to afford 1.7 g of the title compound.

MS (ES+): m/z 525.01 [MH+].

Benzyl{[trans-4-(8-amino-1-iodoimidazo[1,5-a]pyrazin-3-yl)cyclohexyl]methyl}carbamate

A solution of benzyl{[trans-4-(8-chloro-1-iodoimidazo[1,5-a]pyrazin-3-yl)cyclohexyl]methyl}carbamate(1.70 g, 0.00324 mol) in IPA (30 mL) was cooled to −78° C., treated witha stream of ammonia gas over 3 min. and then heated at 110° C. in a Parrvessel overnight. The reaction solution was concentrated in vacuo andresidue washed with water to afford 1.37 g of desired product. ¹H NMR(400 MHz, CDCl₃): δ=1.08-1.17 (m, 2H), 1.88 (m, 1H), 1.71-1.81 (m, 2H),1.91-1.94 (m, 2H), 2.00-2.04 (m, 2H), 2.90 (m, 1H), 3.13 (t, J=6.4 Hz,2H), 4.86 (br, 1H), 5.11 (s, 2H), 5.76 (br, 2H), 7.00 (d, J=5.2 Hz, 1H),7.22 (d, J=5.2 Hz, 1H), 7.31-7.37 (m, 5H). MS (ES+): m/z 5.7.36 [MH+].

Benzyl4-{[(3-chloropyrazin-2-yl)methyl]carbamoyl}piperidine-1-carboxylate

A solution of C-(3-Chloropyrazin-2-yl)methylamine bis-hydrochloride(2.00 g, 0.0107 mol) and N,N-diisopropylethylamine (2.2 g, 0.017 mol) inDCM (27.0 mL) was treated with andN-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (3.2 g,0.017 mol), 1-hydroxybenzotriazole (1.5 g, 0.011 mol) and1-[(benzyloxy)carbonyl]-4-piperidine carboxylic acid (3.8 g, 0.014 mol).The mixture was stirred at rt overnight then diluted with DCM (30 mL),washed with sat. NaHCO₃ (20 mL) and brine (20 mL), then dried overNa₂SO₄ and concentrated in vacuo. The crude material thus obtained waschromatographed over silica gel eluting with EtOAc:hexane 1:1 yielding3.38 g of the title compound. ¹H NMR (400 MHz, CDCl₃): δ 1.68-1.78 (m,2H), 1.91-1.94 (m, 2H), 2.44 (m, 1H), 2.89-2.92 (m, 2H), 4.24-4.26 (m,2H), 4.70 (d, J=4.8 Hz, 2H), 5.14 (s, 2H), 6.85 (br, 1H), 7.30-7.37 (m,5H), 8.34 (d, J=2.8 Hz, 1H), 8.45 (d, J=2.8 Hz, 1H). MS (ES+): m/z389.17 [MH+].

Benzyl 4-(8-chloroimidazo[1,5-a]pyrazin-3-yl)piperidine-1-carboxylate

To a suspension of benzyl4-{[(3-chloropyrazin-2-yl)methyl]carbamoyl}piperidine-1-carboxylate(0.100 g, 0.220 mmol) in EtOAc (0.9 mL) and DMF (0.068 mL) at 0° C. wasslowly added POCl₃ (0.082 mL, 0.88 mmol). After stirring at rt for anhour the mixture was cooled to 0° C. then treated with solid NaHCO₃. Themixture was stirred for 20 min at rt, diluted with water and extractedwith EtOAc (3×20 mL). The combined extracts were washed with water (2×30mL) and brine (30 mL), then dried over Na₂SO₄, and concentrated in vacuoto yield 2.07 g of desired product. ¹H NMR (400 MHz, CDCl₃): δ 1.98-2.04(m, 4H), 3.03-3.20 (m, 3H), 4.30-4.33 (m, 2H), 5.16 (s, 2H), 7.33 (d,J=5.2 Hz, 1H), 7.35-7.38 (m, 5H), 7.26 (d, J=4.4 Hz, 1H), 7.79 (s, 1H).MS (ES+): m/z 371.22 [MH+].

Benzyl4-(8-chloro-1-iodoimidazo[1,5-a]pyrazin-3-yl)piperidine-1-carboxylate

To a solution of benzyl4-(8-chloroimidazo[1,5-a]pyrazin-3-yl)piperidine-1-carboxylate (1.31 g,0.00354 mol) in DMF (0.6 mL) was added NIS (1.6 g, 0.0071 mol). Thereaction mixture was left to stir at 55° C. for 20 h. then the mixturewas diluted with EtOAc (20 mL), washed with water (2×40 mL) and brine,then dried over Na₂SO₄ and concentrated in vacuo. The crude reactionmixture was chromatographed over silica gel eluting withhexane→hexane:EtOAc 1:1 yielding 1.63 g of desired product. ¹H NMR (400MHz, CDCl₃): δ 1.95-2.04 (m, 4H), 3.02-3.15 (m, 3H), 4.29-4.32 (m, 2H),5.15 (s, 2H), 7.32 (d, J=5.2 Hz, 1H), 7.34-7.37 (m, 5H), 7.66 (d, J=5.2Hz, 1H). MS (ES+): m/z 497.03 [MH+].

Benzyl4-(8-amino-1-iodoimidazo[1,5-a]pyrazin-3-yl)piperidine-1-carboxylate

A mixture of benzyl4-(8-chloro-1-iodoimidazo[1,5-a]pyrazin-3-yl)piperidine-1-carboxylate(0.500 g, 0.00101 mol) in IPA (20 mL) was cooled to at −78° C. andtreated with a stream of ammonia gas over 3 minutes. The resultingsolution was heated at 110° C. in a Parr vessel prior to concentrationin vacuo, suspension in DCM and filtration through a bed of Celite. Thefiltrate was concentrated in vacuo to afford 0.504 g of desired product.¹H NMR (400 MHz, CDCl₃): δ 1.88-2.02 (m, 2H), 2.99-3.10 (m, 3H),4.24-4.41 (m, 2H), 5.15 s, 2H), 6.03 (br, 2H), 7.03 (d, J=4.8 Hz, 1H),7.24 (d, J=5.2 Hz, 1H), 7.31-7.40 (m, 5H). MS (ES+): m/z 479.33 [MH+].

1-(2-Trimethylsilylethoxymethyl)-1H-pyrrolo[2,3-b]pyridine

To a suspension of sodium hydride (934 mg, 0.0358 mol) in DMF (57 mL)was added dropwise under N₂, a solution of 1H-pyrrolo[2,3-b]pyridine(3.00 g, 0.0254 mol) in DMF (20 mL). The mixture was stirred at r.t. for45 min. then cooled to 0° C. and treated dropwise with[2-(trimethylsilyl)ethoxy]methyl chloride (6.32 mL, 0.0357 mol). Themixture was stirred at rt for 12 h. then poured into water (10 mL),stirred for 30 min. and extracted with Et2O (4×10 mL). The combinedextracts were washed with brine (20 mL), dried over sodium sulfate, andconcentrated in vacuo to give the crude product which waschromatographed over silica gel eluting with hexane→1:9 Et₂O: hexane toafford 6 g desired product.

N-(2-Trimethylsilyl-1-ethoxymethyl)-2-(tributylstannyl)-1H-pyrrolo[2,3-b]pyridine

To a solution of1-(2-trimethylsilylethoxymethyl)-1H-pyrrolo[2,3-b]pyridine (500 mg,0.0020129 mol) in THF (5 mL) at −10° C. was added a 2.0 M of n-BuLi incyclohexane (1.2 mL). After 10 min at −10° C., the mixture was cooled to−20° C. and tributyltin chloride (0.65 mL, 0.0024 mol) was added. Themixture was stirred at rt for 1 h, the poured into a 5% aqueous ammoniumchloride (20 mL), extracted with EtOAc (3×20 mL) and the combinedextracts dried over anhydrous MgSO₄ and concentrated in vacuo. Thematerial thus obtained was chromatographed over silica gel eluting with1:9 EtOAc:hexane to afford 0.7 g of the title compound. ¹H NMR (400 MHzDMSO-d6) δ 0.01 (s, 9H), 0.10 (s, 2H), 0.92-0.94 (m, 9H), 1.14-1.27 (m,6H), 1.37-1.46 (m, 6H), 1.60-1.72 (m, 6H), 3.48-3.52 (m, 2H), 5.71 (s,2H), 6.74 (s, 1H), 7.16-7.19 (m, 1H), 8.02 (dd, J=1.6, 7.6 Hz, 1H) and8.31 (dd, J=1.6, 4.4 Hz, 1H).

3-Cyclobutyl-1-[1-(2-trimethylsilylethoxymethyl)-1H-pyrrolo[2,3-b]pyridin-2-yl]imidazo[1,5-a]pyrazin-8-amine

A mixture ofN-(2-trimethylsilyl-1-ethoxymethyl)-2-(tributylstannyl)-1H-pyrrolo[2,3-b]pyridine(110 mg, 0.20 mmol), 3-cyclobutyl-1-iodoimidazo[1,5-a]pyrazin-8-amine(50 mg, 0.1592 mmol) and bis(triphenylphosphine)palladium(II) chloride(10 mg, 0.02 mmol) in ethanol (2 mL) was heated at reflux for 48 h. Themixture was then cooled to rt, filtered through a pad of Celite andconcentrated in vacuo. The residue thus obtained was chromatographedover silica gel eluting with hex:EtOAc to afford 17.2 mg of the titlecompound. ¹H NMR (400 MHz CDCl₃) δ 0.22 (s, 9H), 0.70 (t, 2H), 1.87-2.19(m, 2H), 2.49-2.64 (m, 4H), 3.37 (t, 2H), 3.81-3.86 (m, 1H), 5.51 (bs,2H), 6.07 (s, 2H), 6.67 (s, 1H), 7.10-7.16 (m, 3H), 7.93 (dd, J=1.6, 8.0Hz, 1H) and 8.41 (dd, J=1.6, 4.8 Hz, 1H). MS (ES+): m/z: 435.21 [MH+].

4-Bromo-2-nitro-N-phenylaniline

A mixture of 1-bromo-4-fluoro-3-nitrobenzene (2270 mg, 10.01 mmol),aniline (3 ml) and DMF (20 ml) was heated at 100° C. under an atmosphereof Nitrogen for 7 h. The mixture was then concentrated in vacuo, and theresidue triturated with heptane (30 ml) to give the desired product. ¹HNMR (400 MHz, CDCl₃) δ=7.11 (d, 1H, J=9.2 Hz), 7.25-7.29 (m, 3H),7.40-7.45 (m, 3H), 8.35 (d, 1H, J=2.4 Hz) and 9.45 (brs, 1H).

4-Bromo-N-methyl-2-nitroaniline

Prepared according to a procedure analogous to that described for4-bromo-2-nitro-N-phenylaniline. ¹H NMR (400 MHz, CDCl₃): δ=3.02 (d, 3H,J=5.2 Hz), 6.76 (d, 1H, J=9.6 Hz), 7.51-7.54 (m, 1H), 8.02 (brs, 1H) and8.32 (d, 1H, J=2.8 Hz). MS (ES+): m/z 231.05 and 233.08-[MH+].

4-Bromo-N-ethyl-2-nitroaniline

Prepared according to a procedure analogous to that described for4-bromo-2-nitro-N-phenylaniline. ¹H NMR (400 MHz, CDCl₃) δ=1.37 (t, 3H,J=7.2 Hz), 3.31-3.37 (m, 2H), 6.76 (d, 1H, J=8.8 Hz), 7.48-7.51 (m, 1H),7.95 (brs, 1H) and 8.31 (d, 1H, J=2.4 Hz). MS (ES+): m/z 245.07 and247.11 [MH+].

N-Benzyl-4-bromo-2-nitroaniline

Prepared according to a procedure analogous to that described for4-bromo-2-nitro-N-phenylaniline. ¹H NMR (400 MHz, CDCl₃) δ=4.54 (d, 2H,J=5.6 Hz), 6.72 (d, 1H, J=9.2 Hz), 7.30-7.40 (m, 5H), 7.44 (ddd, 1H,J=0.4 & 2.4 & 9.2 Hz), 8.34 (d, 1H, J=2.4 Hz) and 8.41 (brs, 1H). MS(ES+): m/z 245.07 and 247.11-[MH+].

4-Bromo-N¹-phenylbenzene-1,2-diamine

Prepared according to a procedure analogous to that described for4-bromo-2-nitro-N-phenylaniline. ¹H NMR (400 MHz, DMSO-d₆) δ=3.80 (brs,2H), 5.07 (br, s, 1H), 6.70-6.75 (m, 2H), 6.82-6.86 (m, 2H), 6.93 (d,1H, J=2.4 Hz), 6.97 (d, 1H, J=8.0 Hz) and 7.17-7.24 (m, 2H). MS (ES+):m/z 263.17 and 265.20 [MH+].

4-Bromo-N¹-methylbenzene-1,2-diamine

A suspension of 4-bromo-N-methyl-2-nitroaniline (5328 mg, 22.04 mmol) inEtOH (100 ml) was treated with SnCl₂.2H₂O (25.61 g, 110.2 mmol) and theresulting mixture heated at 70° C. under an atmosphere of Nitrogen for 5h. The reaction mixture was then cooled to rt and treated with ice-water(50 ml) followed by aqueous NaOH (4 N) until pH>8. This basic mixturewas then extracted with EtOAc (3×150 ml) and the combined extractswashed with brine (3×100 ml), dried over MgSO₄ and concentrated in vacuoto afford the title compound. ¹H NMR (400 MHz, DMSO-d₆) δ ppm=2.68 (s,3H), 4.74 (brs, 3H), 6.27 (d, 1H, J=8.4 Hz), 6.61 (dd, 1H, J=2.0 & 8.4Hz) and 6.66 (d, 1H, J=2.0 Hz). MS (ES+): m/z 201.10 and 203.12-[MH+].

4-Bromo-N¹-ethylbenzene-1,2-diamine

Prepared according to a procedure analogous to that described for4-bromo-N-methylbenzene-1,2-diamine. ¹H NMR (400 MHz, DMSO-d₆,) δppm=1.19 (t, 3H, J=6.8 Hz), 3.01 (quartet, 2H, J=6.8 Hz), 4.46 (brs,1H), 4.81 (brs, 2H), 6.30 (d, 1H, J=8.4 Hz), 6.58 (dd, 1H, J=2.4 & 8.4Hz) and 6.66 (d, 1H, J=2.0 Hz). MS (ES+): m/z 215.07 and 217.16 [MH+].

N¹-Benzyl-4-bromobenzene-1,2-diamine

Prepared according to a procedure analogous to that described for4-bromo-N-methylbenzene-1,2-diamine. ¹H NMR (400 MHz, DMSO-d₆) δppm=3.39 (brs, 2H), 3.61 (brs, 1H), 4.28 (s, 2H), 6.51 (d, 1H, J=8.4Hz), 6.85-6.89 (m, 2H) and 7.27-7.38 (m, 5H). MS (ES+): m/z 277.20 and279.20 [MH+].

1-Benzyl-5-bromo-2-phenyl-1H-benzimidazole

p-TsOH.H₂O (311.7 mg, 1.606 mmol) was added to a DCM (50 ml) solution ofN¹-benzyl-4-bromobenzene-1,2-diamine (4451 mg, 16.06 mmol) and trimethylorthobenzoate (3096 μl, 17.66 mmol) and the resulting mixture wasstirred at rt under an atmosphere of Nitrogen for 40 h. The reactionmixture was then concentrated in vacuo to give a yellow solid which wastriturated with 40% MeOH/water (375 mL), filtered, washed with saturatedNaHCO₃ (20 ml)+H₂O (80 ml) twice and 40% MeOH/H₂O (2×50 ml), and driedto give the title compound. ¹H NMR (400 MHz, DMSO-d₆) δ ppm=5.44 (s,2H), 7.05-7.08 (m, 3H), 7.30-7.36 (m, 4H), 7.44-7.50 (m, 3H), 7.66-7.68(m, 2H) and 7.99 (dd, 1H, J=0.4 & 1.6 Hz). MS (ES+): m/z 363.20 and365.26 [MH+].

5-Bromo-1-methyl-2-phenyl-1H-benzimidazole

Prepared according to a procedure analogous to that described for1-benzyl-5-bromo-2-phenyl-1H-benzimidazole. ¹H NMR (400 MHz, CDCl₃) δppm=3.86 (s, 3H), 7.26-7.29 (m, 1H), 7.42 (dd, 1H, J=2.0 & 8.4 Hz),7.53-7.56 (m, 3H), 7.74-7.76 (m, 2H) and 7.95 (dd, 1H, J=0.4 & 1.6 Hz).MS (ES+): m/z 287.18 and 289.14 [MH+].

5-Bromo-1-ethyl-2-phenyl-1H-benzimidazole

Prepared according to a procedure analogous to that described for1-benzyl-5-bromo-2-phenyl-1H-benzimidazole. ¹H NMR (400 MHz, CDCl₃) δppm=1.46 (t, 3H, J=7.2 Hz), 4.27 (quartet, 2H, J=7.2 Hz), 7.27 (m, 1H),7.30 (dd, 1H, J=0.4 & 8.8 Hz), 7.42 (dd, 1H, J=1.6 & 8.8 Hz), 7.53-7.55(m, 3H), 7.70-7.72 (m, 2H) and 7.96 (dd, 1H, J=0.4 & 1.6 Hz). MS (ES+):m/z 301.18 and 303.11 [MH+].

5-Bromo-1,2-diphenyl-1H-benzimidazole

Prepared according to a procedure analogous to that described for1-benzyl-5-bromo-2-phenyl-1H-benzimidazole. ¹H NMR (400 MHz, CDCl₃):δ=7.11 (dd, 1H, J=0.4 & 8.4 Hz), 7.27-7.39 (m, 6H), 7.48-7.56 (m, 5H)and 8.01 (dd, 1H, J=0.4 & 1.6 Hz). MS (ES+): m/z 349.20 and 351.22[MH+].

1-Methyl-2-phenyl-5-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-1H-benzimidazole

A mixture of 5-bromo-1-methyl-2-phenyl-1H-benzimidazole (616 mg, 2.14mmol), [1,1′-Bis(diphenylphosphino)ferrocene]dichloropalladium(II)complex with dichloromethane (1:1) (52.6 mg, 0.0644 mmol),bis(pinacolato)diboron (667 mg, 2.57 mmol),1,1′-bis(diphenylphosphino)ferrocene (36.8 mg, 0.0644 mmol) and AcOK(638 mg, 6.44 mmol) in 1,4-dioxane (10 ml) was purged with N₂ for 5 min,and was then heated at 100° C. under an atmosphere of Nitrogen for 16 h.The mixture was then treated with saturated NH₄Cl (20 ml), extractedwith EtOAc (3×20 ml) and the combined extracts washed with brine (3×20ml), dried over MgSO₄ and concentrated in vacuo to afford crude productwhich was purified by chromatography over silica gel eluting with 30%(250 ml) and 40% (250 ml) EtOAc/Heptane to give a white solid that wastriturated with 50% EtOAc/Heptane (10 ml) to yield the title compound.¹H NMR (400 MHz, CDCl₃) δ ppm=1.38 (s, 12H), 3.86 (s, 3H), 7.39 (dd, 1H,J=1.2 & 8.0 Hz), 7.50-7.55 (m, 3H), 7.76-7.79 (m, 3H) and 8.29 (d, 1H,J=0.8 Hz). MS (ES+): m/z 335.29 (100) [MH+].

1-Ethyl-2-phenyl-5-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-1H-benzimidazole

Prepared according to a procedure analogous to that described for1-Methyl-2-phenyl-5-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-1H-benzimidazole.¹H NMR (400 MHz, CDCl₃) δ ppm=1.38 (s, 12H), 1.45 (t, 3H, J=7.2 Hz),4.28 (quartet, 2H, J=7.2 Hz), 7.42 (dd, 1H, J=0.8 & 8.0 Hz), 7.51-7.54(m, 3H), 7.71-7.74 (m, 2H), 7.77 (dd, 1H, J=0.8 & 8.0 Hz) and 8.31 (s,1H). MS (ES+): m/z 349.33 [MH+].

1-Benzyl-2-phenyl-5-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-1H-benzimidazole

Prepared according to a procedure analogous to that described for1-Methyl-2-phenyl-5-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-1H-benzimidazole.¹H NMR (400 MHz, CDCl₃) δ ppm=1.36 (s, 12H), 5.45 (s, 2H), 7.05-7.08 (m,1H), 7.21 (dd, 1H, J=0.8 & 8.0 Hz), 7.26-7.31 (m, 3H), 7.44-7.48 (m,3H), 7.66-7.71 (m, 3H) and 8.36 (m, 1H). MS (ES+): m/z 411.42 [MH+].

1,2-Diphenyl-5-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-1H-benzimidazole

Prepared according to a procedure analogous to that described for1-Methyl-2-phenyl-5-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-1H-benzimidazole.¹H NMR (400 MHz, CDCl₃) δ ppm=1.38 (s, 12H), 7.22 (dd, 1H, J=0.8 & 8.0Hz), 7.29-7.35 (m, 5H), 7.47-7.50 (m, 3H), 7.55-7.57 (m, 2H) and 7.71(dd, 1H, J=0.8 & 8.0 Hz), 8.38 (m, 1H). MS (ES+): m/z 397.43 [MH+].

7-Chloro-2-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-1H-indole

A flask containing Ir(Ome)₂(COD)₂ [Inorganic Syntheses (1985), 23,126](850 mg, 0.0013 mol), 4,4′-di-tert-butyl-[2,2′]bipyridinyl (686 mg,0.00256 mol) and4,4,4′,4′,5,5,5′,5′-octamethyl-2,2′-bi-1,3,2-dioxaborolane (15.2 g,0.0600 mol) was evacuated and refilled with Ar (3×), then charged withanhydrous DME (400 mL, 3 mol) and a solution of 7-chloro-1H-indole(0.086 mol) in DME (10 mL). The resulting mixture was stirred under Arfor 16 h then concentrated and chromatographed over silica gel elutingwith 10% EtOAc/Heptane to afford the desired product as a waxy solid ina 96% yield. ¹H NMR (400 MHz, CDCl₃) δ ppm 1.39 (s, 12H), 7.04 (t,J=7.71 Hz, 1H), 7.15 (d, J=2.27 Hz, 1H), 7.21-7.30 (m, 1H), 7.58 (d,J=8.08 Hz, 1H) and 8.72 (br. s., 1H).

4-Methoxy-2-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-1H-indole

Prepared according to a procedure analogous to that described for7-chloro-2-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-1H-indoleusing 4-methoxy-1H-indole.

7-Bromo-4-methoxy-2-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-1H-indole

Prepared according to a procedure analogous to that described for7-chloro-2-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-1H-indoleusing 7-bromo-4-methoxy-1H-indole.

7-Methyl-2-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-1H-indole

Prepared according to a procedure analogous to that described for7-chloro-2-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-1H-indoleusing 7-methyl-1H-indole.

7-Fluoro-2-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-1H-indole

Prepared according to a procedure analogous to that described for7-chloro-2-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-1H-indoleusing 7-fluoro-1H-indole.

4-Methyl-2-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-1H-indole

Prepared according to a procedure analogous to that described for7-chloro-2-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-1H-indoleusing 4-methyl-1H-indole.

4-Methoxy-1-methyl-2-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-1H-indole

Prepared according to a procedure analogous to that described for7-chloro-2-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-1H-indoleusing 4-methoxy-1-methyl-1H-indole.

7-Ethyl-2-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-1H-indole

Prepared according to a procedure analogous to that described for7-chloro-2-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-1H-indoleusing 7-ethyl-1H-indole.

4,7-Dimethoxy-2-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-1H-indole

Prepared according to a procedure analogous to that described for7-chloro-2-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-1H-indoleusing 4,7-dimethoxy-1H-indole.

2-(4,4,5,5-Tetramethyl-[1,3,2]dioxaborolan-2-yl)-1H-indol-4-yl acetate

Prepared according to a procedure analogous to that described for7-chloro-2-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-1H-indoleusing 1H-indol-4-yl acetate.

2-(4,4,5,5-Tetramethyl-[1,3,2]dioxaborolan-2-yl)-1H-indole-4-carboxylicacid, methyl ester

Prepared according to a procedure analogous to that described for7-chloro-2-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-1H-indoleusing 1H-indole-4-carboxylic acid, methyl ester.

7-Methoxy-2-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-benzofuran

Prepared according to a procedure analogous to that described for7-chloro-2-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-1H-indoleusing 7-methoxy-benzofuran.

4,4,5,5-Tetramethyl-2-(3-methyl-benzo[b]thiophen-2-yl)-[1,3,2]dioxaborolane

Prepared according to a procedure analogous to that described for7-chloro-2-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-1H-indoleusing 3-methyl-benzo[b]thiophene.

3-Methyl-2-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-benzofuran

Prepared according to a procedure analogous to that described for7-chloro-2-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-1H-indoleusing 3-methyl-benzofuran.

7-Bromo-2-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-1H-indole

Prepared according to a procedure analogous to that described for7-chloro-2-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-1H-indoleusing 7-bromo-1H-indole.

3-Methyl-2-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-1H-indole

Prepared according to a procedure analogous to that described for7-chloro-2-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-1H-indoleusing 3-methyl-1H-indole.

7-Methyl-2-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-benzofuran

Prepared according to a procedure analogous to that described for7-chloro-2-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-1H-indoleusing 7-methyl-benzofuran.

7-Methoxy-2-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-1H-indole

Prepared according to a procedure analogous to that described for7-chloro-2-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-1H-indoleusing 7-methoxy-1H-indole.

7-Ethoxy-1H-indole

To a stirred solution of 1H-indol-7-ol (500 mg, 3.75 mmol) in acetone(10 mL) at r.t. was added potassium carbonate (3.11 g, 22.5 mmol),followed by iodoethane (0.45 mL, 5.63 mol). The mixture was stirred atr.t. for 16 h then solvent removed under reduced pressure. The crudeproduct thus obtained was purified by chromatography over silica gel toafford 7-ethoxy-1H-indole: ¹H NMR (400 MHz, MeOD) δ ppm 1.51 (t, J=6.95Hz, 3H), 4.22 (q, J=6.91 Hz, 2H), 6.42 (d, J=3.03 Hz, 1H), 6.63 (d,J=7.58 Hz, 1H), 6.92 (t, J=7.83 Hz, 1H), 7.04-7.23 (m, 2H); MS (ES+):m/z 162.20 (MH+).

7-Ethoxy-2-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-1H-indole

Prepared according to a procedure analogous to that described for7-chloro-2-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-1H-indoleusing 7-ethoxy-1H-indole.

7-Isopropoxy-1H-indole

Made according to the procedure described for 7-ethoxy-1H-indole using2-iodopropane.

7-Isopropoxy-2-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-1H-indole

Prepared according to a procedure analogous to that described for7-chloro-2-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-1H-indoleusing 7-isopropoxy-1H-indole.

7-Trifluoromethyl-2-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-1H-indole

To a stirred mixture of 7-trifluoromethyl-1H-indole-2,3-dione (116 mg)in THF (5.00 mL) was added boron trifluoride etherate (0.205 mL, 1.62mmol) followed by sodium borohydride (71.4 mg, 1.88 mmol). The resultingmixture was stirred at −20° C. for 2 hrs, then water (1 mL) was addedand the mixture was stirred at 0° C. for 10 min. The solution wasacidified to pH=1 with 2N HCl, warmed to r.t. and stirred at r.t. for 20min prior to extraction with EtOAc. The extracts were dried overmagnesium sulphate, concentrated in vacuo and the residue purified bychromatography over silica gel eluting with hexane to give7-trifluoromethyl-1H-indole. ¹H NMR (400 MHz, CDCl₃) δ ppm 6.63-6.68(1H, m), 7.20 (1H, t, J=7.71 Hz), 7.30-7.35 (1H, m), 7.47 (1H, d, J=7.33Hz), 7.83 (1H, d, J=8.08 Hz), and 8.56 (1H, br. s.).

7-Trifluoromethyl-2-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-1H-indole

Prepared according to a procedure analogous to that described for7-chloro-2-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-1H-indoleusing 7-trifluoromethyl-1H-indole.

Ethyl N-[2(trifluoromethoxy)phenyl]carbamate

Ethyl chloroformate (4.4 mL, 0.046 mol) was added to a mixture of2-(trifluoromethoxy)aniline (8.25 g, 0.0466 mol), sodium carbonate (15g, 0.14 mol), 1,4-dioxane (70 mL) and water (70 mL) at 0° C. and thereaction mixture stirred at room temperature overnight. The reactionmixture was then washed with ether, acidified (pH 3) and the productextracted into EtOAc (3×40 mL). The combined extracts were washed withwater (40 mL) and brine (40 mL), dried over Na2SO4 and the solventremoved in vacuo to give the desired product in a 84% yield. ¹H NMR (400MHz, CDCl₃): δ 1.33 (t, J=5.2 Hz, 3H), 4.25 (q, J=6.8 Hz, 2H), 6.91 (br,1H), 7.04 (m, 1H), 7.23 (m, 1H), 7.28 (m, 1H) and 8.2 (m, 1H). MS (ES+):m/z 250.12 [MH+].

Ethyl [2-iodo-6-(trifluoromethoxy)phenyl]carbamate

A 1.4 M solution of sec-butyllithium in cyclohexane (3.0 mL) was addeddrop-wise to a solution of ethyl N-[2-(trifluoromethoxy)phenyl]carbamate(0.5000 g, 0.002006 mol) in THF (9 mL) at −70° C. After stirring for 1hour a solution of iodine (0.51 g, 0.002 mol) in THF (1.0 mL) was addeddrop-wise at −70° C. Stirring was continued for another 1 hour then themixture was quenched with saturated ammonium chloride solution. Water(50 mL) was added and the mixture extracted with diethyl ether (3×40mL). The combined organic phases was washed with 40% sodiummeta-bisulfite solution, water and brine, then dried over Na2SO4 and thesolvent removed in vacuo to give the desired product in a 73% yield. ¹HNMR (400 MHz, CDCl₃): δ 1.29-1.36 (m, 3H), 4.21-4.28 (m, 2H), 6.21 (br,1H), 7.05 (t, J=8.0 Hz, 1H), 7.30 (m, 1H) and 7.80 (dd, J=6.8, 1.2 Hz,1H). MS (ES+): m/z 375.78 [MH+].

Ethyl [2-trifluoromethoxy-6-(trimethylsilanylethynylphenyl)]carbamate

A mixture of Pd(PPh3)₂Cl2 (83 mg, 0.00012 mol) and copper (I) iodide (23mg, 0.00012 mol) in triethylamine (44 mL, 0.32 mol) was heated at 40° C.for 20 min then cooled to rt and ethyl[2-iodo-6-(trifluoromethoxy)phenyl]carbamate (4.50 g, 0.0120 mol) wasadded in one portion. The mixture was stirred at room temperature for 30min, then (trimethylsilyl)acetylene (1.6 mL, 0.011 mol) was added andthe mixture stirred for a further 2 hours. The solvent was removed invacuo and the residue was partitioned between water and diethyl ether(60 mL of each). The organic was washed with 1N HCl and brine, thendried over Na2SO4 then the solvent removed in vacuo. The reaction waschromatographed over silica gel eluting with 20% EtOAc/hexane to affordthe desired product in 80% yield. MS (ES+): m/z 345.99 [MH+].

7-Trifluoromethoxy-1H-indole

Sodium ethoxide (0.65 mL, 0.0017 mol, 2.6M) was added to a solution ofethyl [2-trifluoromethoxy-6-(trimethylsilanylethynylphenyl)]carbamate inEtOH (5.0 mL) and the mixture stirred at 72° C. for 14 hours. Thesolvent was removed under reduced pressure and the residue waspartitioned between diethyl ether and water (30 mL of each). The etherphase was washed with brine and dried over Na₂SO₄ yielding the desiredcompound in 59% yield. ¹H NMR (400 MHz, CDCl₃): δ 6.60-6.61 (m, 1H),7.07-7.09 (m, 2H), 7.25 (d, J=5.6 Hz, 1H), 7.55-7.57 (m, 1H) and 8.42(br, 1H). MS (ES+): m/z 202.18 [MH+].

7-Trifluoromethoxy-2-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-1H-indole

Prepared according to a procedure analogous to that described for7-chloro-2-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-1H-indoleusing 7-trifluoromethoxy-1H-indole.

7-Phenyl-1H-indole

To a suspension of 7-bromo-1H-indole (196 mg, 0.00100 mol) in1,4-dioxane (4 mL) and water (1 mL) was added phenylboronic acid (146mg, 0.00120 mol), potassium carbonate (414 mg, 0.00300 mol) and[1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium(II), complexwith dichloromethane (1:1) (82 mg, 0.00010 mol). The flask was evacuatedand refilled with nitrogen, three times then the mixture was heated at100° C. overnight. The mixture was diluted with EtOAc (30 mL), washedwith sat. aq. NaHCO₃ (10 mL) and brine (10 mL), then dried overanhydrous sodium sulfate and the solvent removed in vacuo. The crudematerial was purified by chromatography over silica gel eluting withhexane/EtOAc to give the title compound (180 mg, 93% yield). ¹H NMR(CDCl₃, 400 MHz): δ 6.64 (dd, J=3.0, 2.0 Hz, 1H), 7.18-7.26 (m, 3H),7.41 (t, J=7.5 Hz, 1H), 7.48-7.57 (m, 2H), 7.61-7.70 (m, 3H) and 8.43(br s, 1H) ppm. LC-MS (ES+.): 194 [MH⁺].

7-Phenyl-2-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-1H-indole

Prepared according to a procedure analogous to that described for7-chloro-2-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-1H-indoleusing 7-phenyl-1H-indole.

7-Cyclopropyl-2-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-1H-indole

Prepared according to the procedures described above for7-phenyl-2-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-1H-indoleusing cyclopropylboronic acid in place of phenylboronic acid. ¹H NMR(CDCl₃, 400 MHz): δ 0.75-0.82 (m, 2H), 0.95-1.04 (m, 2H), 2.08 (m, 1H),6.59 (dd, J=3.0, 2.0 Hz, 1H), 6.96 (d, J=7.1 Hz, 1H), 7.06 (t, J=7.6 Hz,1H), 7.25 (m, 1H), 7.52 (d, J=7.8 Hz, 1H) and 8.39 (br s, 1H) ppm. LC-MS(ES, Pos.): 158 [MH⁺].

6-Bromo-7-fluoro-1H-indole

To a solution of 1-bromo-2-fluoro-3-nitrobenzene (2.5 g, 11.3 mmol) inTHF (25 mL) at −50° C. was added vinyl magnesium bromide (34 mL, 34mmol) and the mixture was stirred at −40° C. for 1 h. The reaction wasquenched with saturated ammonium chloride solution and extracted withethyl acetate. The organic layer was washed with brine, dried overanhydrous sodium sulfate and evaporated under reduced pressure to yielda gum, which was purified by column chromatography over silica geleluting with EtOAc/hexane to afford pure 6-bromo-7-fluoro-1H-indole. ¹HNMR (400 MHz, CDCl₃) δ=6.53-6.62 (m, 1H), 7.16-7.25 (m, 2H), 7.29 (d,J=8.34 Hz, 1H) and 8.36 (br. s., 1H); MS (ES+): m/z 214.08 [MH+].

6-Bromo-7-fluoro-1-methyl-1H-indole

To a solution of 6-bromo-7-fluoro-1H-indole (470 mg, 2.19 mmol) in THF(7 mL) at −10° C. was added sodium hydride (175 mg, 4.39 mmol, 60%dispersion) and the mixture was stirred at 0° C. for 30 min. Methyliodide was added at 0° C. and the reaction was allowed to warm to at 10°C. and stirred for 2 h. The reaction was quenched with saturatedammonium chloride and extracted with DCM. The DCM extract was washedwith brine, dried over anhydrous sodium sulfate and evaporated underreduced pressure. The crude product was purified by columnchromatography over silica gel eluting with EtOAc/hexane to afford6-bromo-7-fluoro-1-methyl-1H-indole. ¹H NMR (400 MHz, CDCl₃) δ=3.95 (d,J=2.00 Hz, 1H), 6.42 (t, J=2.78 Hz, 1H), 6.94 (d, J=3.03 Hz, 1H),7.09-7.15 (m, 1H) and 7.20 (d, J=8.34 Hz, 1H); MS (ES+): m/z 228.04[MH+].

7-Fluoro-1-methyl-6-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-1H-indole

To a mixture of 6-bromo-7-fluoro-1-methyl-1H-indole (420 mg, 1.84 mmol),4,4,4′,4′,5,5,5′,5′-octamethyl-2,2′-bi-1,3,2-dioxaborolane (514 mg, 2.02mmol), potassium acetate (542 mg, 5.52 mmol),[1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium(II) complex withdichloromethane (1:1 complex, 150 mg, 0.184 mmol) and1,1′-bis(diphenylphosphino)ferrocene (102 mg, 0.184 mmol) was addeddioxane (10 mL) and the mixture was degassed by bubbling through withnitrogen for 3 min. The reaction mixture was heated at 100° C. overnightthen the dioxane was removed under reduced pressure and the residue wasdissolved in DCM and filtered to remove inorganics. The filtrate wasconcentrated and the crude product was purified by column chromatographyover silica gel eluting with EtOAc/hexane to afford pure7-fluoro-1-methyl-6-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-1H-indole.¹H NMR (400 MHz, CDCl₃) δ=1.41 (s, 12H), 4.02 (d, J=2.02 Hz, 3H), 6.46(t, J=2.65 Hz, 1H), 7.03 (d, J=3.03 Hz, 1H) and 7.28-7.47 (m, 2H); MS(ES+): m/z 276.03 [MH+].

7-Trifluoromethyl-benzo[b]thiophene

To a stirred solution of 2-(trifluoromethyl)benzenethiol (5.000 g, 0.028mol) in acetone (50 mL) was added 2-bromo-1,1-diethoxyethane (6.08 g,0.030 mol) and potassium carbonate (7.757 g, 0.056 mol). The resultingmixture was then stirred at 45° C. for 2 hours prior to removal of thesolvent in vacuo and suspension of the residue in EtOAc. The inorganicsalts were filtered off and the organic phase was concentrated to givecrude product, which was used in next step without further purification.This residue was dissolved in toluene (50 mL), and to this solution wasadded PPA (10 g) and the resulting mixture stirred at 95-100° C. for 2hours. The mixture was allowed to cool to rt, was poured into ice-water,then extracted with EtOAc (3×50 mL). The combined extracts were washedwith aqueous sodium bicarbonate followed by brine, then dried overanhydrous sodium sulfate and evaporated under reduced pressure to yieldan oil. This was purified by column chromatography over silica geleluting with hexane to give 7-trifluoromethyl-benzo[b]thiophene. ¹H NMR(400 MHz, MeOD) δ ppm 7.49-7.57 (m, 2H), 7.70 (d, J=7.33 Hz, 1H), 7.74(d, J=5.56 Hz, 1H) and 8.10 (d, J=8.08 Hz, 1H).

7-Trifluoromethylbenzo[b]thiophene-2-boronic acid

To a solution of 7-trifluoromethyl-benzo[b]thiophene (0.52 g, 0.0026mol) in THF (30 mL) at −78° C. was added 2.5 M of n-BuLi in hexane (1.4mL). The reaction was then slowly warmed up to −30° C. over 30 min. andstirred at this temperature for 10 min prior to recooling to −78° C. andtreatment with triisopropyl borate (0.7255 g, 0.0038 mol). The reactionwas then slowly warmed up to 0° C. then was quenched with saturatedammonium chloride and the solvent removed in vacuo. To the residue wasadded aqueous sodium hydroxide (10 mL, 2N solution) followed by water(30 mL) then this mixture was extracted with DCM. The aqueous solutionwas acidified using dilute sulfuric acid (2N solution), filtered and theresidue dried in vacuo to yield7-trifluoromethylbenzo[b]thiophen-2-boronic acid. ¹H NMR (400 MHz, MeOD)δ ppm 7.55 (1H, t, J=7.45 Hz), 7.75 (1H, d, J=7.07 Hz), 8.02 (1H, s) and8.17 (1H, d, J=7.83 Hz).

N-Methylindole-6-boronic acid

A mixture of indole-6-boronic acid (0.100 g, 0.615 mmol), sodium hydride(0.07 g, 20 mmol) and THF (5 mL, 60 mmol) was stirred at rt for 20 min.then methyl iodide (100 uL, 20 mmol) was added and the mixture wasallowed ro stir at rt for 3 hours. The reaction was quenched with sat.NH₄Cl solution, washed with brine and dried over Na₂SO4, then thesolvent was removed in vacuo. The crude product was purified bychromatography over silica gel eluting with 1:9 EtOAc/hexane and 1%MeOH, yielding the desired product. ¹H NMR (400 MHz, CDCl₃) δ ppm 3.99(s, 3H), 6.58 (m, 1H). 7.23 (m, 1H), 7.81 (m, 1H), 8.08 (m, 1H) and 8.34(m, 1H). MS (ES+): m/z 176.15 [MH+].

4-Bromo-3-methyl-2-nitrophenol

To a solution of 3-methyl-2-nitrophenol (2.0 g, 13.06 mmol) in aceticacid (40 mL) was added bromine (0.70 mL, 13.71 mmol) and the mixture wasstirred at RT for 5 h. The reaction was poured in to ice water and theyellow precipitate formed was filtered and washed with water and driedin vacuo to yield 4-bromo-3-methyl-2-nitrophenol. ¹H NMR (400 MHz,CDCl₃) δ=2.61 (s, 3H), 2.62 (s, 5H), 6.92 (d, J=8.84 Hz, 1H), 7.66 (d,J=9.09 Hz, 1H) and 9.28 (s, 1H); MS (ES+): m/z 215.00 [M-17].

1-Bromo-4-methoxy-2-methyl-3-nitrobenzene

To a solution of 4-bromo-3-methyl-2-nitrophenol (2.200 g, 9.48 mmol) inacetone (35 mL) was added potassium carbonate (3.276 g, 23.70 mmol) andmethyl iodide (1.47 mL, 23.70 mmol) and the mixture was heated to refluxfor 4 h. The reaction was cooled to rt, filtered and the filtrate wasevaporated under reduced pressure to afford the crude product.Purification of the crude product by column chromatography over silicagel eluting with EtOAc/hexane afforded pure1-bromo-4-methoxy-2-methyl-3-nitrobenzene as pale yellow solid. ¹H NMR(400 MHz, CDCl₃) δ=2.33 (s, 2H), 3.87 (s, 3H), 6.78 (d, J=8.84 Hz, 1H)and 7.58 (d, J=8.84 Hz, 1H); MS (ES+): m/z 247.26 [MH+].

1-[(E)-2-(6-bromo-3-methoxy-2-nitrophenyl)vinyl]pyrrolidine

To a solution of 1-bromo-4-methoxy-2-methyl-3-nitrobenzene (1.400 g,5.68 mmol) and 1,1-dimethoxy-N,N-dimethylmethanamine (0.884 mL, 6.657mmol) in DMF (10.0 mL) was added pyrrolidine (0.555 mL, 6.656 mmol) andthe mixture was heated to at 110° C. for 4 h. The DMF was removed andthe residue was recrystallized from DCM:methanol (1:6) mixture to afford1-[(E)-2-(6-bromo-3-methoxy-2-nitrophenyl)vinyl]pyrrolidine.

4-Bromo-7-methoxy-1H-indole

To a solution of1-[(E)-2-(6-bromo-3-methoxy-2-nitrophenyl)vinyl]pyrrolidine (1.3 g, 3.97mmol) in THF (6 mL) and methanol (6 mL) was added Raney Ni (≈500 mg)followed by hydrazine (0.19 mL). (CAUTION: Exothermic reaction withvigorous gas evolution). Hydrazine (0.19 mL) was added again, two times,after 30 min and 1 h. The reaction was stirred at 45° C. for 2 h,filtered through a pad of celite. The filtrate was concentrated in vacuoand the residue purified by chromatography over silica gel eluting withEtOAc/hexane to afford pure 4-bromo-7-methoxy-1H-indole. ¹H NMR (400MHz, CDCl₃) δ=3.94 (s, 3H), 6.52 (d, J=8.08 Hz, 1H), 6.56 (dd, J=3.16,2.40 Hz, 1H), 7.17 (d, J=8.08 Hz, 1H), 7.22 (t, J=2.78 Hz, 1H) and 8.47(br. s., 1H); MS (ES+): m/z 226.12 [MH+].

2-Phenyl-5-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-1,3-benzothiazole

A stirred solution of 5-bromo-2-phenylbenzothiazole (0.500 g, 0.00172mol), bis(pinacolato)diboron (0.508 g, 0.00200 mol),1,3-bis(2,6-diisopropylphenyl)imidazol-2-ylidene hydrochloride (0.044 g,0.10 mmol), Pd(OAc)2 (0.019 g, 0.086 mmol) and AcOK (0.423 g, 0.00431mol) in anhydrous THF (9.78 mL, 0.121 mol) was heated at 72° C. underArgon for 29 h. The mixture was filtered through a multi-layered pad ofanhydrous sodium sulfate, silica gel and celite and the filtrate wasconcentrated in vacuo and the solids triturated multiple times withhexanes to give the title compound. ¹H NMR (400 MHz, CHLOROFORM-d) δppm=1.39 (s, 12H), 7.49-7.56 (m, 3H), 7.83 (dd, J=8.08, 1.01 Hz, 1H),7.92 (d, J=7.33 Hz, 1H), 8.12-8.18 (m, 2H) and 8.60 (s, 1H); MS (ES+):m/z 337.91 [MH+].

4-(Methoxycarbonyl)-4-methylcyclohexanecarboxylic acid

N,N-Diisopropylamine (1.18 mL, 8.35 mmol) was added dropwise to a 2Msolution of ^(n)butyllithium (4.18 mL, 8.4 mmol) at −78° C. undernitrogen. After 15 min at this temperature the solution was raised toand held at 0° C. for 15 min prior to re-cooling to −78° C. andtreatment with a solution of 4-(methoxycarbonyl)cyclohexanecarboxylicacid (0.62 g, 3.34 mmol) in THF (8 mL). After 30 min., iodomethane (0.31mL, 5 mmol) was added dropwise and the mixture was allowed to warm to rtover 2 hr. The mixture was cooled to at 0° C., quenched with 2 N HCl (10mL) then was extracted with EtOAc (2×10 mL), washed with brine (3×15mL), and dried over anhydrous magnesium sulfate. Concentration of thecombined organic extracts afforded a yellow solid. NMR (CDCl₃)consistent with crude, desired product.

Methyltrans-4-{[(2,5-dioxopyrrolidin-1-yl)oxy]carbonyl}cyclohexanecarboxylate

A solution of N-hydroxysuccinimide (6.18 g, 0.0537 mol) andtrans-4-(methoxycarbonyl)cyclohexanecarboxylic acid (10.00 g, 0.05370mol) in THF (100.00 mL) was charged with (N,N-dicyclohexylcarbodiimide(11.08 g, 0.0537 mol) in THF (16 mL). This reaction was stirred at rtfor an additional 16 h then stirred at 45° C. for 1 h. The reactionmixture was filtered while still warm through a fritted funnel. The cakewas washed with 3 more portions of THF and the filtrate was concentratedin vacuo and was crystallized from i-PrOH (300 mL) and filtered througha fritted funnel resulting in 11.8 g, (78% yield) of the title compoundas a white crystals. ¹H NMR (400 MHz, CDCl₃) δ ppm 1.45-1.69 (m, 4H),2.07-2.16 (m, 2H), 2.18-2.28 (m, 2H), 2.29-2.39 (m, 1H), 2.59-2.71 (m,1H) 2.84 (br. s., 4H) and 3.68 (s, 3H); MS (ES+): m/z 284.09 [MH+].

Methyltrans-4-{[(3-amino-5-oxo-4,5-dihydro-1,2,4-triazin-6-yl)methyl]carbamoyl}cyclohexanecarboxylate

A solution of 3-amino-6-(aminomethyl)-1,2,4-triazin-5(4H)-one [J.Heterocyclic Chem., (1984), 21 (3), 697](2.00 g, 0.0113 mol) in H₂O(60.0 mL, 3.33 mol) was cooled to 0° C. and drop wise charged with 1.00M of NaHCO₃ in H₂O (22.5 mL) and allowed to warm to rt. This mixture wascharged with methyltrans-4-{[(2,5-dioxopyrrolidin-1-yl)oxy]carbonyl}cyclohexanecarboxylate(3.8 g, 0.012 mol) in 1:1 THF/MeCN (40 mL). After 30 min a precipitatebegan to form in the reaction. This was allowed to stir at rt for anadditional 16 h and was filtered through a fritted funnel and washedwith H₂O (2×), diethyl ether (2×), and dried in vacuo resulting in thetitle compound 2.92 g, (84% yield) as an off-white solid. ¹H NMR (400MHz, DMSO-d₆) δ ppm 1.24-1.55 (m, 4H), 1.83 (s, 2H), 1.98 (d, J=10.61Hz, 2H), 2.27 (s, 2H), 3.64 (s, 3H), 4.10 (d, J=5.81 Hz, 2H), 6.81 (br.s., 2H), 7.91 (t, J=5.56 Hz, 1H) and 11.98 (br. s., 1H); MS (ES+): m/z310.05 [MH+].

Methyltrans-4-(2-amino-4-oxo-3,4-dihydroimidazo[5,1-f][1,2,4]triazin-7-yl)cyclohexanecarboxylate

A solution of methyltrans-4-{[(3-amino-5-oxo-4,5-dihydro-1,2,4-triazin-6-yl)methyl]carbamoyl}cyclohexanecarboxylate(2.00 g, 0.00646 mol) in 1,2-dichloroethane (130 mL) was charged withPOCl₃ (4.2 mL, 0.045 mol) and heated to reflux for 3 h. The reactionmixture was concentrated in vacuo then partitioned between EtOAc andsat. NaHCO₃ and separated. The aqueous was re-extracted with EtOAc (3×)and the combined organic fractions were dried over Na₂SO₄, filtered, andconcentrated in vacuo resulting in 1.43 g, (76% yield) of the titlecompound as an off-white solid. ¹H NMR (400 MHz, DMSO-d₆) δ ppm 1.43 (q,J=11.79 Hz, 2H), 1.61 (q, J=12.55 Hz, 2H), 1.85-2.11 (m, 4H), 2.38 (t,J=11.87 Hz, 1H), 2.98 (t, J=11.75 Hz, 1H), 3.61 (s, 3H), 6.17 (br. s.,2H), 7.49 (s, 1H) and 10.90 (br. s., 1H); MS (ES+): m/z 292.25 [MH+].

Methyltrans-4-(2-amino-5-iodo-4-oxo-3,4-dihydroimidazo[5,1-f][1,2,4]triazin-7-yl)cyclohexanecarboxylate

A solution of methyltrans-4-(2-amino-4-oxo-3,4-dihydroimidazo[5,1-f][1,2,4]triazin-7-yl)cyclohexanecarboxylate(0.200 g, 0.000686 mol) and N-iodosuccinimide (0.278 g, 0.00124 mol) inanhydrous DMF (4.0 mL) was stirred at rt for 48 h. The reaction wasconcentrated in vacuo then partitioned between H₂O and EtOAc. Theaqueous material was re-extracted with EtOAc (3×) and the combinedorganic fractions were washed with H₂O (2×), Na₂S₂O₃ (2×) and brine(1×). The aqueous was re-extracted with CHCl₃ and combined with theEtOAc fractions dried over Na₂SO₄, filtered and concentrated in vacuoresulting in 229 mg, (79.9% yield) of the title compound as a lightorange solid. ¹H NMR (400 MHz, DMSO-d₆) δ ppm 1.34-1.65 (m, 4H),1.88-2.06 (m, 4H), 2.33-2.45 (m, 1H), 2.91-3.01 (m, 1H), 3.61 (s, 3H),6.17 (s, 2H) and 10.82 (br. s., 1H); MS (ES+): m/z 417.82 [MH+].

Methyltrans-4-(5-iodo-4-oxo-3,4-dihydroimidazo[5,1-f][1,2,4]triazin-7-yl)cyclohexanecarboxylate

A solution of methyltrans-4-(2-amino-5-iodo-4-oxo-3,4-dihydroimidazo[5,1-f][1,2,4]triazin-7-yl)cyclohexanecarboxylate(0.880 g, 0.00211 mol) in anhydrous THF (74 mL) and DMF (13.2 mL) wascharged with tert-butyl nitrite (1.2 mL, 0.010 mol) and stirred at rtfor 2 h. The reaction was concentrated in vacuo and was purified bychromatography over silica gel [eluting with 5% MeOH in CHCl₃]resultingin 570 mg, (67% yield) of the title compound as a pale orange solid. (¹HNMR (400 MHz, DMSO-d₆) δ ppm 1.40-1.54 (m, 2H), 1.56-1.69 (m, 2H),1.92-2.06 (m, 4H), 2.36-2.46 (m, 1H), 3.02-3.14 (m, 1H), 3.61 (s, 3H),7.89 (d, J=3.28 Hz, 1H) and 11.79 (br. s., 1H); MS (ES+): m/z 402.86[MH+].

Methyltrans-4-(4-amino-5-iodoimidazo[5,1-f][1,2,4]triazin-7-yl)cyclohexanecarboxylate

A solution of 1H-1,2,4-triazole (0.881 g, 0.0128 mol) in pyridine (3.00mL) was charged with POCl₃ (0.396 mL, 0.00425 mol) and stirred at rt for15 min. To this mixture was drop wise added methyltrans-4-(5-iodo-4-oxo-3,4-dihydroimidazo[5,1-f][1,2,4]triazin-7-yl)cyclohexanecarboxylate(0.570 g, 0.00142 mol) in pyridine (6.00 mL) and stirred at rt for anadditional 2.45 h. The reaction was quenched with excess 2 M of NH₃ ini-PrOH (40.00 mL) at 0° C. and allowed to stir at rt for an additional 3h. The reaction was concentrated in vacuo and partitioned between EtOAcand sat. NaHCO₃ and separated. The aqueous was washed with EtOAc (3×)and the combined organic fractions were washed with brine (1×). Theaqueous was re-extracted with CHCl₃ (3×) and the organic was added tothe EtOAc fractions. The combined organic fractions were dried overNa₂SO₄, filtered and concentrated in vacuo. The crude brown/red solidwas purified by chromatography over silica gel [eluting with 5% MeOH inCHCl₃]resulting in 438 mg, (76% yield) of the title compound as a lightyellow solid. ¹H NMR (400 MHz, DMSO-d₆) δ ppm 1.39-1.54 (m, 2H),1.55-1.71 (m, 2H), 1.92-2.07 (m, 4H), 2.35-2.46 (m, 1H), 3.06-3.19 (m,1H), 3.61 (s, 3H), 6.77 (br. s., 1H) 7.86 (s, 1H) and 8.44 (br. s., 1H);MS (ES+): m/z 401.85 [MH+].

1-Chloro-2-[(2,2-diethoxyethyl)thio]benzene

To a solution of 2-chlorobenzenethiol (5.0 g, 34.5 mmol) in acetone (35mL) was added 2-bromo-1,1-diethoxyethane (7.15 g, 36.3 mmol) followed bypotassium carbonate (9.55 g, 69.1 mmol). The mixture was heated atreflux for 3 h. then cooled to rt, filtered and the filtrate evaporatedunder reduced pressure to yield the crude product. This material waspurified by chromatography over silica gel eluting with ethyl acetate inhexanes (0→2%) to afford pure1-chloro-2-(2,2-diethoxyethylsulfanyl)benzene (7.3, 80%). ¹H NMR (400MHz, CDCl₃) δ=1.20 (t, J=7.07 Hz, 6H), 3.15 (d, J=5.56 Hz, 2H),3.51-3.61 (m, 2H), 3.63-3.74 (m, 2H), 4.69 (t, J=5.56 Hz, 1H), 7.12 (td,J=7.58, 1.52 Hz, 1H), 7.20 (td, J=7.58, 1.52 Hz, 1H), 7.36 (dd, J=7.83,1.52 Hz, 1H), 7.39 (dd, J=8.08, 1.52 Hz, 1H); MS (ES+): m/z 187.17[M-74].

7-Chlorobenzo[b]thiophene

To a solution of 1-chloro-2-(2,2-diethoxyethylsulfanyl)benzene (3.95 g,15.14 mmol) in toluene (40 mL) was added polyphosphoric acid (15 g,137.5 mmol).

The mixture was heated at reflux for 4 h. then was poured in to icewater, stirred for min and extracted with toluene. The combined tolueneextracts were washed with aqueous sodium bicarbonate followed by brine,dried over anhydrous sodium sulfate and evaporated under reducedpressure to yield the crude product. This material was purified bychromatography over silica gel eluting with hexane to afford pure7-chlorobenzo[b]thiophene (1.72 g, 67.5%). ¹H NMR (400 MHz, CDCl₃)δ=7.13-7.30 (m, 3H), 7.38 (d, J=5.31 Hz, 1H), 7.62 (dd, J=7.33, 1.52 Hz,1H); MS (ES+): m/z 169.06 [MH+].

7-Chlorobenzo[b]thiophene-2-boronic acid

To a solution of 7-chlorobenzo[b]thiophene (1.0 g, 5.92 mmol) in THF (25mL) at −78° C. was added ^(n)butyllithium (7.41 mL, 11.8 mmol, 1.6 Msolution). The reaction was allowed to warm to −30° C. then was cooledback to −78° C. and triisopropyl borate (2.23 g, 11.8 mmol) was added.The mixture was allowed to warm to 0° C., saturated ammonium chlorideadded and the organic phase separated off and concentrated in vacuo. Tothe residue was added aqueous sodium hydroxide (10 mL, 2N solution)followed by water (30 mL) and the mixture was washed with DCM. Theaqueous phase was acidified with 2N sulfuric acid, and the resultingprecipitate isolated by filtration and dried under vacuum to yield7-chlorobenzo[b]thiophene-2-boronic acid (1.21 g, 96%) as white solid.¹H NMR (400 MHz, CDCl₃) δ=7.41 (t, J=7.70 Hz, 1H), 7.50 (d, J=7.70 Hz,1H), 7.91 (d, J=7.70 Hz, 1H), 8.03 (s, 1H), 8.63 (s, 2H); MS (ES+): m/z211.86 [M+].

7-(methylthio)-1H-indole

To a solution of 7-bromo-1H-indole (3.0 g, 15.3 mmol) in THF (60 mL) at−78° C. was added ^(t)BuLi (1.7 M, 33.8 mL, 57.4 mmol) and the mixturewas allowed to warm to 0° C. The reaction was re-cooled to −78° C. and asolution of dimethyl disulfide (2.0 mL, 22.9 mmol) was added and thereaction was allowed to warm to 0° C. The reaction was quenched withsaturated ammonium chloride and extracted with ethyl acetate. Theorganic layer was washed with brine, dried over anhydrous sodium sulfateand evaporated under reduced pressure to yield the crude product. Thismaterial was purified by chromatography over silica gel eluting withethyl acetate in hexanes (0-2%) to afford pure 7-(methylthio)-1H-indole(1.4 g, 55%). ¹H NMR (400 MHz, CDCl₃) δ=2.50 (s, 3H), 6.58 (dd, J=3.03,2.02 Hz, 1H), 7.09 (t, J=7.58 Hz, 1H), 7.18-7.31 (m, 2H), 7.56 (d,J=7.83 Hz, 1H), 8.45 (br. s., 1H); MS (ES+): m/z 164.15 [MH+].

7-(Methylsulfonyl)-1H-indole

To a solution of 7-(methylthio)-1H-indole (1.1 g, 6.7 mmol) in DCM (25ml) at −40° C. was added m-chloroperbenzoic acid (3.02 g, 13.4 mmol) andthe reaction was stirred at −40° C. for 30 min. The reaction mixture wasthen quenched with saturated sodium bicarbonate and extracted with DCM.The DCM extracts was washed with water, brine, dried over anhydroussodium sulfate and evaporated under reduced pressure to yield the crudeproduct. This material was purified by chromatography over silica geleluting with hexanes (0->10%) to afford pure7-(methylsulfonyl)-1H-indole (987 mg, 75%). ¹H NMR (400 MHz, CDCl₃)δ=3.12 (s, 1H), 6.66 (d, J=2.53 Hz, 1H), 7.24 (t, J=7.71 Hz, 1H), 7.35(d, J=1.77 Hz, 1H), 7.68 (d, J=7.07 Hz, 1H), 7.90 (d, J=7.83 Hz, 1H),9.68 (br. s., 1H); MS (ES+): m/z 196.08 [MH+].

Methyl trans-4-cyanocyclohexanecarboxylate

Chlorosulfonyl isocyanate (1.0 mL, 0.012 mol) was added to a solution oftrans-4-(methoxycarbonyl)cyclohexanecarboxylic acid (2.00 g, 0.0107 mol)in DCM cooled to 0° C. The resulting solution was heated at reflux for15 minutes and then cooled 0° C. and treated dropwise with DMF. Themixture was stirred at room temperature overnight then poured onto icewater and the organic phase separated and washed with a saturatedsolution of sodium bicarbonate. The solvent was removed in vacuo and thecrude material was taken up in ethyl acetate, washed with 1N aq. NaOH(10 mL) and the ethyl acetate removed in vacuo. The resulting crudeproduct was used in subsequent steps without further purification. ¹HNMR (400 MHz, CHLOROFORM-d) δ ppm 1.36-1.70 (4H, m), 2.01-2.18 (4H, m),2.24-2.54 (2H, m) and 3.68 (3H, s).

Trans-4-cyanocyclohexanecarboxylic acid

To a solution of methyl trans-4-cyanocyclohexanecarboxylate (996 mg,5.96 mmol) in THF (37 mL) was added a solution of 0.5 M lithiumhydroxide in water (20 mL). The mixture was stirred overnight then theTHF was removed in vacuo and the residual aqueous solution acidified topH 4. The resulting mixture was extracted with ether (2×30 mL), EtOAc(2×30 mL) and CHCl₃ (2×30 mL) then the combined extracts, dried overanhydrous sodium sulfate and concentrated in vacuo. This material wastaken to the next step without any purification. ¹H NMR (400 MHz,CHLOROFORM-d) δ ppm 1.43-1.73 (4H, m), 2.05-2.22 (4H, m) and 2.36-2.59(2H, m).

2-[Trans-4-(8-chloroimidazo[1,5-a]pyrazin-3-yl)cyclohexyl]propan-2-ol

A solution of methyltrans-4-(8-chloroimidazo[1,5-a]pyrazin-3-yl)cyclohexanecarboxylate (4.0g, 0.014 mol) in toluene (300 mL) and THF (70 mL) was cooled to 0° C.and treated with a 3.0 M solution of methylmagnesium bromide in ether(14 mL) maintaining the temperature at 0° C. The mixture was stirred atrt for 1.5 hours then cooled to 0° C. and an additional 3 eq of 3.0 M ofmethylmagnesium bromide in ether was added. The mixture was stirred atrt for 15 minutes then cooled to 0° C. and quenched with 1:1 NH₄Cl sat.:H₂O (50 mL total volume). The organic layer was separated and theaqueous layer was extracted with EtOAc (3×30 mL). The combined organiclayers were dried over sodium sulfate and concentrated in vacuo and thecrude product thus obtained, chromatographed over silica gel elutingwith EtOAc to afford desired2-[trans-4-(8-chloroimidazo[1,5-a]pyrazin-3-yl)cyclohexyl]propan-2-ol.¹H NMR (400 MHz, CHLOROFORM-d) δ ppm 1.14-1.39 (m, 8H), 1.41-1.60 (m,1H), 1.77-1.98 (m, 2H), 2.01-2.20 (m, 4H), 2.78-3.06 (m, 1H), 7.35 (d,J=5.05 Hz, 1H), 7.64 (d, J=5.05 Hz, 1H) and 7.83 (s, 1H).

Example 1

3-Cyclobutyl-1-(1H-indol-5-yl)imidazo[1,5-a]pyrazin-8-amine

A dry mixture of 8-amino-3-cyclobutyl-1-iodoimidazo[3,4-a]pyrazine (30mg, 0.096 mmol), cesium carbonate (38 mg, 0.117 mmol) and5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-indole (26 mg, 0.107mmol) was purged with Argon 3 times prior to the addition oftetrakistriphenylphosphino palladium (0) (6 mg, 0.005 mmol). The mixturewas purged twice more and then treated with a degassed mixture ofDME:water (5:1, 2 mL). The resulting solution was degassed twice moreand then heated at 80° C. overnight. The resulting reaction mixture wasconcentrated in vacuo, the residue dissolved in 1:1 MeCN:MeOH (1.5 mL)and purified by mass directed preparative HPLC to afford3-cyclobutyl-1-(1H-indol-5-yl)imidazo[1,5-a]pyrazin-8-amine. ¹H NMR (400MHz, DMSO-d₆) δ ppm 1.82-1.92 (1H, m) 1.95-2.08 (1H, m) 2.32-2.41 (4H,m) 3.82-3.93 (1H, m) 5.91 (2H, br. s.) 6.45 (1H, d, J=3.03 Hz) 6.90 (1H,d, J=5.05 Hz) 7.26 (1H, dd, J=8.34, 1.52 Hz) 7.34 (1H, d, J=5.05 Hz)7.35-7.39 (1H, m) 7.45 (1H, d, J=8.34 Hz) 7.64-7.68 (1H, m) 11.20 (1H,br. s.); MS (ES+): m/z 304.15 [MH+]. HPLC: t_(R) 6.18 min (XTerra C18 5uM, 4.6×15 mm, A: MeCN & B:10 mmol NH₄OAc in 0.05% HOAc/aq., methodPolar15).

Example 2

3-Cyclobutyl-1-(1H-indol-2-yl)imidazo[1,5-a]pyrazin-8-amine

Prepared as described above for EXAMPLE 1 using1-(tert-butoxycarbonyl)-1H-indole-2-boronic acid in place of5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-indole. The reactionconditions used effected significant cleavage of theN-(tert-butoxycarbamoyl) functionality. MS (ES+): m/z 304.10 [MH+].

Example 3

3-Cyclobutyl-1-(5-fluoro-1H-indol-2-yl)imidazo[1,5-a]pyrazin-8-amine

Prepared as described above for EXAMPLE 1 using1-(tert-butoxycarbonyl)-5-fluoro-1H-indole-2-boronic acid in place of5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-indole. The reactionconditions used effected significant cleavage of theN-(tert-butoxycarbamoyl) functionality. MS (ES+): m/z 322.06 [MH+].

Example 4

1-(1-Benzothien-5-yl)-3-cyclobutylimidazo[1,5-a]pyrazin-8-amine

Prepared as described above for EXAMPLE 1 using2-(1-benzothiophen-5-yl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane inplace of 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-indole. MS(ES+): m/z 321.10 [MH+].

Example 5

3-Cyclobutyl-1-(5-methyl-1H-indol-2-yl)imidazo[1,5-a]pyrazin-8-amine

Prepared as described above for EXAMPLE 1 using1-(tert-butoxycarbonyl)-5-methyl-1H-indole-2-boronic acid in place of5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-indole. MS (ES+): m/z318.05 [MH+].

Example 6

3-Cyclobutyl-1-(6-methyl-1H-indol-2-yl)imidazo[1,5-a]pyrazin-8-amine

Prepared as described above for EXAMPLE 1 using1-(tert-butoxycarbonyl)-6-methyl-1H-indole-2-boronic acid in place of5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-indole. MS (ES+): m/z318.05 [MH+].

Example 7

3-Cyclobutyl-1-(1H-indol-6-yl)imidazo[1,5-a]pyrazin-8-amine

A mixture of 6-bromo-1H-indole (2 g, 10.00 mmol),4,4,4′,4′,5,5,5′,5′-octamethyl-2,2′-bi-1,3,2-dioxaborolane (2.00 g, 7.87mmol) and potassium acetate (3.0 g, 31.00 mmol) was degassed threetimes, treated with (1,1′-bis(diphenylphosphino)ferrocene) palladiumdichloride (0.20 g, 0.28 mmol) and degassed twice more.1,2-dimethoxyethane (28 mL) was added and the mixture was heated at 75°C. overnight. The cooled reaction mixture was then diluted with water,extracted with EtOAc and the extracts washed with water and brine, thendried over magnesium sulphate, and concentrated in vacuo to afford abrown/black semi-solid. This was triturated with ether to afford a brownpowder, which was identified by LCMS to be desired indole-6-boronicacid, pinacol ester. ¹H NMR (400 MHz, CHLOROFORM-d) δ ppm 1.37 (s, 12H),6.54-6.58 (m, 1H), 7.26-7.28 (m, 1H), 7.55 (dd, J=7.83, 1.01 Hz, 1H),7.62-7.68 (m, 1H), 7.90 (s, 1H), 8.19 (br. s., 1H); MS (ES+): m/z 244.25[MH⁺]; HPLC: t_(R)=3.52 min (OpenLynx, polar_(—)5 min).

This material was used in place of5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-indole under theconditions described in EXAMPLE 1 to afford3-cyclobutyl-1-(1H-indol-6-yl)imidazo[1,5-a]pyrazin-8-amine. MS (ES+):m/z 304.15 [MH+].

Example 8

1-(1H-Benzimidazol-2-yl)-3-cyclobutylimidazo[1,5-a]pyrazin-8-amine

3-Cyclobutyl-1-iodoimidazo[1,5-a]pyrazin-8-amine (500 mg, 2 mmol) andtetrakis(triphenylphosphine)palladium(0) (100 mg, 0.1 mmol) was degasseddry three times then treated with methanol (20 mL) andN,N-diisopropylethylamine (0.7 mL, 4.0 mmol) and the mixture heated at70° C. under an atmosphere of carbon monoxide, with intermittentbubbling of this gas under the surface of the reaction mixture. After 3dheating with extensive bubbling through of the solution with carbonmonoxide and some addition of fresh catalyst after day 2, TLC (10%MeOH/DCM) indicated the reaction to be complete. The reaction mixturewas diluted with water, extracted with DCM and the extracts washed withwater and brine, then dried over magnesium sulphate, and concentrated invacuo to afford an orange solid which was recrystallised fromacetonitrile to afford methyl8-amino-3-cyclobutylimidazo[1,5-a]pyrazine-1-carboxylate. ¹H NMR (400MHz, CHLOROFORM-d) δ ppm 1.97-2.06 (m, 1H), 2.10-2.26 (m, 1H), 2.43-2.54(m, 2H), 2.53-2.68 (m, 2H), 3.78 (dd, J=9.09, 8.08 Hz, 1H), 4.01 (s,3H), 7.08 (d, J=4.80 Hz, 1H), 7.22 (d, J=4.80 Hz, 1H), 7.38 (br. s.,1H), 7.69 (br. s., 1H).

A suspension of 1,2-phenylenediamine (60 mg, 0.6 mmol) in toluene (2.0mL) was treated with a 2M solution of trimethylaluminum in toluene (0.5mL) effecting the formation of a pink solution. After 5 min thissolution was treated with solid methyl8-amino-3-cyclobutylimidazo[1,5-a]pyrazine-1-carboxylate (30 mg, 0.1mmol) and the mixture heated at 120° C. for 30 min then stirred at rtovernight. The mixture was then partitioned between 2M NaOH (10 mL) &EtOAc (10 mL) and stirred for 15 min. The organic layer was separatedand the aqueous layer extracted further with EtOAc (3×10 mL). Thecombined organics were washed with brine, dried and concentrated invacuo to give ˜85% pure8-amino-N-(2-aminophenyl)-3-cyclobutylimidazo[1,5-a]pyrazine-1-carboxamidewhich was used without purification.

A solution of8-amino-N-(2-aminophenyl)-3-cyclobutylimidazo[1,5-a]pyrazine-1-carboxamide(40.0 mg, 0.124 mmol) in acetic acid (1.2 mL) was microwaved at 120° C.for 10 min (300W). The resulting solution was purified mass directedpreparative HPLC to afford1-(1H-benzimidazol-2-yl)-3-cyclobutylimidazo[1,5-a]pyrazin-8-amine. ¹HNMR (400 MHz, DMSO-d6) δ ppm 1.92-2.05 (m, 1H) 2.07-2.21 (m, 1H)2.53-2.59 (m, 4H) 3.91-4.06 (m, 1H) 7.08 (d, J=4.80 Hz, 1H) 7.16-7.26(m, 2H) 7.38 (d, J=4.80 Hz, 1H) 7.44 (br. s., 1H) 7.55 (d, J=8.08 Hz,1H) 7.62 (d, J=6.82 Hz, 1H) 10.49 (br. s., 1H) 12.76 (s, 1H); MS (ES+):m/z 305.15 [MH⁺].

Example 9

1-(1,3-Benzoxazol-5-yl)-3-cyclobutylimidazo[1,5-a]pyrazin-8-amine

A mixture of 5-chlorobenzoxazole (0.129 g, 0.84 mmol),4,4,4′,4′,5,5,5′,5′-octamethyl-2,2′-bi-1,3,2-dioxaborolane (0.4956 g,1.95 mmol), potassium acetate (0.41 g, 4.2 mmol),1,3-bis(2,6-diisopropylphenyl)imidazol-2-ylidene hydrochloride (43 mg,0.10 mmol) and palladium acetate (11 mg, 0.05 mmol) was degassed,treated with tetrahydrofuran (10 mL) and the resulting mixture heated at80° C. overnight. The mixture was diluted with water (100 mL), acidifiedto pH 6 and extracted with EtOAc (3×40 mL). The extracts were washedwith water, dried and concentrated in vacuo. The residue so obtained waspurified by chromatography over silica gel eluting with DCM to 10%MeCN/DCM to afford 1,3-benzoxazole-5-boronic acid, pinacol ester. ¹H NMR(400 MHz, CHLOROFORM-d) δ ppm 1.37-1.39 (m, 12H) 7.59 (d, J=8.34 Hz, 1H)7.86 (dd, J=8.08, 1.01 Hz, 1H) 8.10 (s, 1H) 8.26 (s, 1H); MS (ES+): m/z246.23 [MH⁺].

This material was used in place of5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-indole under theconditions described in example 1 to afford1-(1,3-benzoxazol-5-yl)-3-cyclobutylimidazo[1,5-a]pyrazin-8-amine MS(ES+): m/z 306.16 [MH+].

Example 10

{trans-4-[8-Amino-1-(1H-indol-2-yl)imidazo[1,5-a]pyrazin-3-yl]cyclohexyl}methanol

Prepared according to the procedure described in EXAMPLE 2 usingtrans-[4-(8-amino-1-iodoimidazo[1,5-a]pyrazin-3-yl)cyclohexyl]methanolin place of 8-amino-3-cyclobutyl-1-iodoimidazo[3,4-a]pyrazine. ¹H NMR(DMSO-d6, 400 MHz) δ 1.12-1.23 (m,), 1.38-1.54 (m, 1H); 1.58-1.78 (m,2H); 1.82-1.92 (m, 2H); 1.96-2.06 (m, 2H); 3.03-3.16 (m, 1H); 3.29 (t,J=5.6 Hz, 2H); 4.46 (t, J=5.3 Hz, 1H); 6.45 (brs, 2H); 6.63 (d, J=1.38Hz, 1H); 7.02 (t, J=7.50 Hz, 1H); 7.06 (d, J=4.99 Hz, 1H); 7.12 (t,J=7.52, 1H), 7.46 (d, J=8.02 Hz, 1H), 7.58 (d, J=7.83 Hz, 1H), 7.66 (d,J=5.06 Hz, 1H), 11.43 (s, 1H); MS (ES+): m/z 362.07 (100) [MH+], HPLC:tR=1.97 min (MicromassZQ, polar_(—)5 min).

Example 11

{cis-3-[8-Amino-1-(1H-indol-2-yl)imidazo[1,5-a]pyrazin-3-yl]cyclobutyl}methanol

Prepared according to the procedure described in EXAMPLE 2 using[3-(8-chloro-1-iodoimidazo[1,5-a]pyrazin-3-yl)cyclobutyl]methanol inplace of 8-amino-3-cyclobutyl-1-iodoimidazo[3,4-a]pyrazine. MS (ES+):m/z 334.10 [MH+].

Example 12

cis-3-[8-Amino-1-(1H-indol-2-yl)imidazo[1,5-a]pyrazin-3-yl]cyclobutanol

Prepared according to the procedure described in EXAMPLE 2 using3-(8-amino-1-iodoimidazo[1,5-a]pyrazin-3-yl)cyclobutanol in place of8-amino-3-cyclobutyl-1-iodoimidazo[3,4-a]pyrazine. MS (ES+): m/z 320.03[MH+].

Example 13

3-[cis-3-(4-Acetylpiperazin-1-yl)cyclobutyl]-1-(1H-indol-2-yl)imidazo[1,5-a]pyrazin-8-amine

Prepared according to the procedure described in EXAMPLE 2 using1-{4-[3-(8-amino-1-iodoimidazo[1,5-a]pyrazin-3-yl)cyclobutyl]piperazin-1-yl}ethanonein place of 8-amino-3-cyclobutyl-1-iodoimidazo[3,4-a]pyrazine. MS (ES+):m/z 430.08 [MH+].

Example 14

{trans-4-[8-Amino-1-(1H-indol-5-yl)imidazo[1,5-a]pyrazin-3-yl]cyclohexyl}methanol

Prepared according to the procedure described in EXAMPLE 1 usingtrans-[4-(8-amino-1-iodoimidazo[1,5-a]pyrazin-3-yl)cyclohexyl]methanolin place of 8-amino-3-cyclobutyl-1-iodoimidazo[3,4-a]pyrazine. MS (ES+):m/z 362.07 [MH+].

Example 15

1-(1H-Indol-2-yl)-3-[cis-3-(4-methylpiperazin-1-yl)cyclobutyl]imidazo[1,5-a]pyrazin-8-amine

Prepared according to the procedure described in EXAMPLE 2 using1-iodo-3-[3-(4-methyl-piperazin-1-yl)cyclobutyl]imidazo[1,5-a]pyrazin-8-ylaminein place of 8-amino-3-cyclobutyl-1-iodoimidazo[3,4-a]pyrazine. MS (ES+):m/z 402.10 [MH+].

Example 16

7-Cyclobutyl-5-(1H-indol-5-yl)imidazo[5,1-f][1,2,4]triazin-4-amine

Prepared according to the procedure described in EXAMPLE 1 using7-cyclobutyl-5-iodoimidazo[5,1-f][1,2,4]triazin-4-ylamine in place of8-amino-3-cyclobutyl-1-iodoimidazo[3,4-a]pyrazine. MS (ES+): m/z 305.16[MH+].

Example 17

7-Cyclobutyl-5-(1H-indol-2-yl)imidazo[5,1-f][1,2,4]triazin-4-amine

Prepared according to the procedure described in EXAMPLE 2 using7-cyclobutyl-5-iodoimidazo[5,1-f][1,2,4]triazin-4-ylamine in place of8-amino-3-cyclobutyl-1-iodoimidazo[3,4-a]pyrazine. MS (ES+): m/z 305.07[MH⁺].

Example 18

7-Cyclobutyl-5-(1H-indol-6-yl)imidazo[5,1-f][1,2,4]triazin-4-amine

Prepared according to the procedure described in EXAMPLE 7 using7-cyclobutyl-5-iodoimidazo[5,1-f][1,2,4]triazin-4-ylamine in place of8-amino-3-cyclobutyl-1-iodoimidazo[3,4-a]pyrazine. MS (ES+): m/z 305.07[MH+].

Example 19

7-Cyclohexyl-5-(1H-indol-2-yl)imidazo[5,1-f][1,2,4]triazin-4-amine

Prepared according to the procedure described in EXAMPLE 2 using7-cyclohexyl-5-iodoimidazo[5,1-f][1,2,4]triazin-4-amine in place of8-amino-3-cyclobutyl-1-iodoimidazo[3,4-a]pyrazine. ¹H NMR (400MHz-DMSO-d6) δ 1.40-1.54 (m, 4H), 1.72-1.82 (m, 2H), 1.87-1.92 (m, 2H),2.02-2.09 (m, 2H) 3.31-3.38 (m, 1H) 6.26 (bs, 2H) 6.73-6.74 (m, 1H),7.13-7.17 (m, 1H), 7.22-7.25 (m, 1H), 7.44 (d, J=8.0 Hz, 1H) 7.64 (d,J=8.0 Hz, 1H), 7.91 (s, 1H), 9.18 (s, 1H). MS (ES+): m/z: 333.16 (100)[MH+]. HPLC: t_(R)=3.46 min (OpenLynx: polar_(—)5 min).

Example 20

A mixture of{trans-4-[8-amino-1-(1H-indol-2-yl)imidazo[1,5-a]pyrazin-3-yl]cyclohexyl}methanol(400 mg, 0.001 mol), phthalimide (211.7 mg, 0.001439 mol), andtriphenylphosphine resin (2.14 mmol/g loading; 1.03 g, 0.00221 mol;Argonaut) in THF (22 mL, 0.27 mol; Aldrich) was placed under nitrogenatmosphere and charged dropwise with diisopropyl azodicarboxylate (290.9mg, 0.001439 mol). After 16 h, the resin was filtered off, washed withchloroform (5×20 mL) and the filtrate concentrated in vacuo to yield anorange oil which was chromatographed over silica gel eluting withchloroform→5% MeOH/chloroform to afford the title compound. ¹H NMR(CDCl₃, 400 MHz): δ 7.90-7.85 (m, 2H), 7.77-7.70 (m, 2H), 7.64 (m, 1H),7.43 (dd, J=8.0, 0.8 Hz, 1H), 7.27-7.15 (m, 2H), 7.14 (m, 1H), 7.09 (d,J=4.8 Hz, 1H), 6.77 (br s, 1H), 3.64 (d, J=6.4 Hz, 2H), 2.91 (m, 1H),2.09 (m, 2H), 2.25-1.90 (m, 4H), 1.80 (ddd, J=13.2, 12.4, 2.4 Hz, 2H),1.27 (ddd, J=13.2, 12.4, 2.4 Hz, 2H). MS (ES+): m/z 491.09 [MH+].

Example 21

1-{trans-4-[8-Amino-1-(1H-indol-2-yl)imidazo[1,5-a]pyrazin-3-yl]cyclohexyl}methanamine

A solution of benzyl{[trans-4-(8-amino-1-(1H-indol-2-yl)imidazo[1,5-a]pyrazin-3-yl)cyclohexyl]methyl}carbamate(0.163 g, 0.330 mmol) in cone. HCl (5 ml) was stirred at rt overnight.The reaction mixture was diluted with H₂O (20 mL), washed with Et₂O (30mL), then basified with 1N NaOH (aq) and extracted with DCM (3×20 mL).The combined extracts were washed with water then dried over Na₂SO₄ andconcentrated in vacuo To afford 0.085 g of desired compound. MS (ES+):m/z 361.30 [MH+].

Example 22

N-({trans-4-[8-Amino-1-(1H-indol-2-yl)imidazo[1,5-a]pyrazin-3-yl]cyclohexyl}methyl)acetamide

To a suspension of1-{trans-4-[8-amino-1-(1H-indol-2-yl)imidazo[1,5-a]pyrazin-3-yl]cyclohexyl}methanamine(100.00 mg, 0.27 mmol), N-(3-dimethylaminopropyl)-N¹-ethylcarbodiimidehydrochloride (0.0798 g, 0.416 mmol), N,N-diisopropylethylamine (0.097mL, 0.55 mmol), 1-hydroxbenzotriaxole Hydrate (0.0425 g, 0.277 mmol),and DMF (600 uL) in DCM (5 mL) was added AcOH (24 uL). The mixture wasstirred at rt for 3 h under an atmosphere of nitrogen then diluted withDCM (20 mL), washed with saturated NaHCO₃ (aq) (2×25 mL) and brine (2×25mL), then dried over Na₂SO₄, filtered and concentrated in vacuo. Theresidue was chromatographed over silica gel eluting with DCM→2% 2M NH₃in MeOH/DCM to afford 0.02 g of the title compound. MS (ES+): m/z 403.31[MH+]. ¹H NMR (400 MHz, CDCl₃): δ 1.12-1.31 (m, 3H), 1.79-1.86 (m, 2H),1.94-1.97 (m, 2H), 2.02 (s, 3H), 2.04-2.09 (m, 2H), 2.91 (m, 1H), 3.20(t, J=6.4 Hz, 2H), 5.51 (br, 1H), 5.66 (br, 2H), 6.79 (s, 1H), 7.10-7.16(m, 2H), 7.20-7.25 (m, 2H), 7.43 (d, J=8.4 Hz, 1H), 7.44 (d, J=7.6 Hz,1H), 9.07 (br, 1H).

Example 23

N-({4-[8-amino-1-(1H-indol-2-yl)imidazo[1,5-a]pyrazin-3-yl]cyclohexyl}methyl)methanesulfonamide

Methanesulfonyl chloride (4.40 μL, 0.057 mmol was added to a mixture of1-{trans-4-[8-amino-1-(1H-indol-2-yl)imidazo[1,5-a]pyrazin-3-yl]cyclohexyl}methanamine(20.5 mg, 0.057 mol) and PS-DIEA (3.90 mmol/g loading; 60 mg, 0.2 mmol)in DCM (1.14 mL). The reaction mixture was stirred for 30 min at r.t.for 18 h. The crude reaction mixture was then concentrated and residuepurified by mass directed preparative HPLC to afford 4 mg of desiredproduct. MS (ES+): m/z 439.10 (100) [MH+]. ¹H NMR (CD3OD, 400 MHz): δ8.24 (br s, 2H), 7.61 (m, 2H), 7.46 (dd, J=8.4, 0.8 Hz, 1H), 7.19 (ddd,J=7.2, 1.2, 1.2 Hz, 1H), 7.08 (ddd, J=7.2, 1.2, 1.2 Hz, 1H), 6.75 (d,J=0.8 Hz, 1H), 3.14 (m, 1H), 2.07 (m, 4H), 1.85 (m, 2H), 1.64 (m, 1H),1.26 (m, 2H).

Example 24

Benzyl4-[8-amino-1-(1H-indol-2-yl)imidazo[1,5-a]pyrazin-3-yl]piperidine-1-carboxylate

A mixture of benzyl4-(8-amino-1-iodoimidazo[1,5-a]pyrazin-3-yl)piperidine-1-carboxylate(1.149 g, 0.002191 mol), 1-(tert-butoxycarbonyl)-1H-indole-2-boronicacid (0.629 g, 0.00241 mol), 1,2-dimethoxyethane (9.3 mL), water (1.8mL) and cesium carbonate (1.43 g, 0.00438 mol) was degassed three timesand then treated with tetrakis(triphenyl phosphine)palladium(0) (200 mg,0.0002 mol). The mixture was once more degassed and then heated at 100°C. overnight. The resulting reaction mixture was diluted with EtOAc (30mL) then washed with water (2×30 mL) and brine, dried over Na₂SO₄ andconcentrated in vacuo. The crude product was chromatographed over silicagel eluting with hexane→EtOAc:hexane 1:1:0.05 2M NH₃/MeOH to afford thedesired product. ¹H NMR (400 MHz, CDCl₃): δ 2.02-2.06 (m, 4H), 3.03-3.17(m, 3H), 4.29-4.33 (m, 2H), 5.16 (s, 2H), 5.66 (br, 2H), 6.79-6.80 (m,1H), 7.11-7.16 (m, 2H), 7.20-7.25 (m, 2H), 7.31-7.45 (m, 5H), 7.44 (m,1H), 7.64 (d, J=7.6 Hz, 1H), 8.96 (br, 1H). MS (ES+): m/z 467.12 [MH+].

Example 25

1-(1H-Indol-2-yl)-3-piperidin-4-ylimidazo[1,5-a]pyrazin-8-amine

A solution of benzyl4-[8-amino-1-(1H-indol-2-yl)imidazo[1,5-a]pyrazin-3-yl]piperidine-1-carboxylate(3.61 g, 0.00774 mol) in conc. HCl (100 ml) was stirred at rt overnight.The mixture was then diluted with water (200 mL), washed with Et₂O (2×30mL) then the aqueous layer concentrated in vacuo yielding 2.62 g ofdesired product as the trihydrochloride salt. ¹H NMR (400 MHz, MeOD): δ2.19-2.32 (m, 4H), 3.26-3.30 (m, 2H), 3.53-3.36 (m, 2H), 3.70 (m, 1H),7.06 (d, J=5.6 Hz, 1H), 7.10-7.14 (m, 1H), 7.23-7.26 (m, 2H), 7.50-7.52(m, 1H), 7.67 (m, 1H), 7.93 (m, 1H). MS (ES+): m/z 333.27 [MH+].

Example 26

4-[8-Amino-1-(1H-indol-2-yl)imidazo[1,5-a]pyrazin-3-yl]piperidine-1-carbaldehyde

To a solution of1-(1H-Indol-2-yl)-3-piperidin-4-ylimidazo[1,5-a]pyrazin-8-aminehydrochloride (30.00 mg, 0.0068 mmol) in DCM (0.5 mL, 0.008 mol) wasadded N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride(0.0195 g, 0.102 mmol), N,N-diisopropylethylamine (0.047 mL),1-hydroxbenzotriaxole hydrate (0.0104 g, 0.0679 mmol) and formic acid(4.7 mg, 0.10 mmol). The reaction was stirred at rt overnight thendiluted with DCM, washed with saturated NaHCO₃ (2×25 mL) and brine(2×25), then dried over Na₂SO₄ and concentrated in vacuo. The materialthus isolated was crystallized from EtOAc to afford 10.6 mg of desiredproduct. ¹H NMR (400 MHz, CDCl₃): δ 2.04-2.12 (m, 4H), 2.99-3.00 (m,1H), 3.27-3.32 (m, 2H), 3.85 (m, 1H), 4.49 (m, 1H), 5.70 (br, 2H), 6.80(s, 1H), 7.13-7.24 (m, 4H), 7.45 (d, J=8.4 Hz, 1H), 7.65 (d, J=8.0 Hz,1H), 8.10 (s, 1H), 8.97 (br, 1H). MS (ES+): m/z 361.16 [MH+].

Example 27

3-[1-(1H-Indol-3-ylcarbonyl)piperidin-4-yl]-1-(1H-indol-2-yl)imidazo[1,5-a]pyrazin-8-amine

Prepared according to the procedure described above for EXAMPLE 26,except using indole-3-carboxylic acid in place of formic acid. MS (ES+):m/z 476.18 [MH+].

Example 28

3-(1-Acetylpiperidin-4-yl)-1-(1H-indol-2-yl)imidazo[1,5-a]pyrazin-8-amine

Prepared according to the procedure described above for EXAMPLE 26,except using acetic acid in place of formic acid. MS (ES+): m/z 375.17[MH+].

Example 29

3-[1-(4-Methoxybenzoyl)piperidin-4-yl]-1-(1H-indol-2-yl)imidazo[1,5-a]pyrazin-8-amine

Prepared according to the procedure described above for EXAMPLE 26,except using 4-methoxybenzoic acid in place of formic acid. MS (ES+):m/z 467.27 [MH+].

Example 30

3-[1-(4-Bromobenzoyl)piperidin-4-yl]-1-(1H-indol-2-yl)imidazo[1,5-a]pyrazin-8-amine

Prepared according to the procedure described above for EXAMPLE 26,except using 4-methoxybenzoic acid in place of formic acid. MS (ES+):m/z 515.17 & 517.17 [MH+].

Example 31

1-(1H-Indol-2-yl-3-[1-(methoxyacetyl)piperidin-4-yl]imidazo[1,5-a]pyrazin-8-amine

Prepared according to the procedure described above for EXAMPLE 26,except using 2-methoxyacetic acid in place of formic acid. MS (ES+): m/z405.10 [MH+].

Example 32

3-[1-(Cyclopentylcarbonyl)piperidin-4-yl]-1-(1H-indol-2-yl)imidazo[1,5-a]pyrazin-8-amine

Prepared according to the procedure described above for EXAMPLE 26,except using cyclopentanecarboxylic acid in place of formic acid. MS(ES+): m/z 429.07 [MH+].

Example 33

3-{1-[(2,5-Dimethyl-1H-pyrrol-3-yl)carbonyl]piperidin-4-yl}-1-(1H-indol-2-yl)imidazo[1,5-a]pyrazin-8-amine

Prepared according to the procedure described above for EXAMPLE 26,except using 2,5-dimethylpyrrolecarboxylic acid in place of formic acid.MS (ES+): m/z 454.19 [MH+].

Example 34

3-{1-[4-(Dimethylamino)butanoyl]piperidin-4-yl}-1-(1H-indol-2-yl)imidazo[1,5-a]pyrazin-8-amine

Prepared according to the procedure described above for EXAMPLE 26,except using 4-(dimethylamino)butanoic acid in place of formic acid. MS(ES+): m/z 446.22 [MH+].

Example 35

3-{1-[4-(Dimethylamino)phenacyl]piperidin-4-yl}-1-(1H-indol-2-yl)imidazo[1,5-a]pyrazin-8-amine

Prepared according to the procedure described above for EXAMPLE 26,except using 4-(dimethylamino)phenylacetic acid in place of formic acid.MS (ES+): m/z 480.22 [MH+].

Example 36

3-{1-[4-(Dimethylamino)benzoyl]piperidin-4-yl}-1-(1H-indol-2-yl)imidazo[1,5-a]pyrazin-8-amine

Prepared according to the procedure described above for EXAMPLE 26,except using 4-(dimethylamino)benzoic acid in place of formic acid. MS(ES+): m/z 480.22 [MH+].

Example 37

3-[1-(Cyclohexylcarbonyl)piperidin-4-yl]-1-(1H-indol-2-yl)imidazo[1,5-a]pyrazin-8-amine

Prepared according to the procedure described above for EXAMPLE 26,except using cyclohexanecarboxylic acid in place of formic acid. MS(ES+): m/z 443.20 [MH+].

Example 38

3-[1-(Cyclopropylcarbonyl)piperidin-4-yl]-1-(1H-indol-2-yl)imidazo[1,5-a]pyrazin-8-amine

Prepared according to the procedure described above for EXAMPLE 26,except using cyclopropanecarboxylic acid in place of formic acid. MS(ES+): m/z 401.19 [MH+].

Example 39

1-(1H-Indol-2-yl)-3-[1-(2-thienylcarbonyl)piperidin-4-yl]imidazo[1,5-a]pyrazin-8-amine

Prepared according to the procedure described above for EXAMPLE 26,except using thiophene-2-carboxylic acid in place of formic acid. MS(ES+): m/z 443.22 [MH+].

Example 40

3-[1-(1H-Indol-3-ylacetyl)piperidin-4-yl]-1-(1H-indol-2-yl)imidazo[1,5-a]pyrazin-8-amine

Prepared according to the procedure described above for EXAMPLE 26,except using indole-3-acetic acid in place of formic acid. MS (ES+): m/z490.10 [MH+].

Example 41

1-(1H-Indol-2-yl)-3-{1-[(3-methoxyphenoxy)acetyl]piperidin-4-yl}imidazo[1,5-a]pyrazin-8-amine

Prepared according to the procedure described above for EXAMPLE 26,except using (3-methoxyphenoxy)acetic acid in place of formic acid. MS(ES+): m/z 497.11 [MH+].

Example 42

3-[1-(1,3-Benzodioxol-5-ylcarbonyl)piperidin-4-yl]-1-(1H-indol-2-yl)imidazo[1,5-a]pyrazin-8-amine

Prepared according to the procedure described above for EXAMPLE 26,except using 1,3-benzodioxole-5-carboxylic acid in place of formic acid.MS (ES+): m/z 481.05 [MH+].

Example 43

1-(1H-Indol-2-yl)-3-{1-[(1-methyl-1H-indazol-3-yl)carbonyl]piperidin-4-yl}imidazo[1,5-a]pyrazin-8-amine

Prepared according to the procedure described above for EXAMPLE 26,except using 1-methyl-1H-indazole-3-carboxylic acid in place of formicacid. MS (ES+): m/z 491.04 [MH+].

Example 44

1-(1H-Indol-2-yl)-3-{1-[(3-methoxyphenyl)acetyl]piperidin-4-yl}imidazo[1,5-a]pyrazin-8-amine

Prepared according to the procedure described above for EXAMPLE 26,except using 3-methoxyphenylacetic acid in place of formic acid. MS(ES+): m/z 481.09 [MH+].

Example 45

3-[1-(1-Benzothien-3-ylcarbonyl)piperidin-4-yl]-1-iodoimidazo[1,5-a]pyrazin-8-amine

Prepared according to the procedure described above for EXAMPLE 26,except using benzothiophene-3-carboxylic acid in place of formic acid.MS (ES+): m/z 493.01 [MH+].

Example 46

3-[1-(1,3-Benzothiazol-6-ylcarbonyl)piperidin-4-yl]-1-iodoimidazo[1,5-a]pyrazin-8-amine

Prepared according to the procedure described above for EXAMPLE 26,except using benzothiazole-6-carboxylic acid in place of formic acid. MS(ES+): m/z 494.01 [MH+].

Example 47

1-(1H-Indol-2-yl)-3-{1-[(2-methylcyclohexa-2,5-dien-1-yl)carbonyl]piperidin-4-yl}imidazo[1,5-a]pyrazin-8-amine

Prepared according to the procedure described above for EXAMPLE 26,except using 2-methylcyclohexa-2,5-diene-1-carboxylic acid in place offormic acid. MS (ES+): m/z 453.08 [MH+].

Example 48

1-(1H-Indol-2-yl)-3-[1-(isoquinolin-1-ylcarbonyl)piperidin-4-yl]imidazo[1,5-a]pyrazin-8-amine

Prepared according to the procedure described above for EXAMPLE 26,except using isoquinoline-1-carboxylic acid in place of formic acid. MS(ES+): m/z 488.01 [MH+].

Example 49

1-(1H-Indol-2-yl)-3-{1-[(pyridin-4-ylthio)acetyl]piperidin-4-yl}imidazo[1,5-a]pyrazin-8-amine

Prepared according to the procedure described above for EXAMPLE 26,except using (pyridin-4-ylthio)acetic acid in place of formic acid. MS(ES+): m/z 484.04 [MH+].

Example 50

1-(1H-Indol-2-yl)-3-[1-(pyridin-3-ylacetyl)piperidin-4-yl]imidazo[1,5-a]pyrazin-8-amine

Prepared according to the procedure described above for EXAMPLE 26,except using pyridin-3-ylacetic acid in place of formic acid. MS (ES+):m/z 452.07 [MH+].

Example 51

4-(8-Amino-1-(1H-indol-2-yl)imidazo[1,5-a]pyrazin-3-yl)-N,N-dimethylpiperidine-1-carboxamide

A mixture of1-(1H-indol-2-yl)-3-piperidin-4-ylimidazo[1,5-a]pyrazin-8-aminehydrochloride (30.0 mg, 0.0679 mmol), N,N-diisopropylethylamine (59.1μL, 0.340 mmol) and DMF (1.00 mL) was treated with N,N-dimethylcarbamoylchloride (6.23 μL, 0.0679 mmol) and stirred at rt for 1 h prior tosemi-preparative HPLC to afford the isolated title compound. ¹H NMR (400MHz, CD₃OD) ppm: 8.32 (br. s., 1H), 7.59-7.66 (m, 2H), 7.46 (d, 1H,J=8.3 Hz), 7.15-7.22 (m, 1H), 7.01-7.10 (m, 2H), 6.74 (s, 1H), 3.82 (d,2H, J=12.6 Hz), 3.34-3.42 (m, 1H), 2.97-3.09 (m, 2H), 2.87 (s, 6H),1.95-2.09 (m, 4H); MS (ES+): m/z 404.14 [MH+].

Example 52

Methyl4-(8-amino-1-(1H-indol-2-yl)imidazo[1,5-a]pyrazin-3-yl)piperidine-1-carboxylate

A mixture of1-(1H-indol-2-yl)-3-piperidin-4-ylimidazo[1,5-a]pyrazin-8-aminehydrochloride (30.0 mg, 0.0679 mmol), N,N-diisopropylethylamine (59.1μL, 0.340 mmol) and DMF (1.00 mL) was treated with methyl chloroformate(5.25 μL, 0.0679 mmol) and stirred at rt for 1 h prior tosemi-preparative HPLC to afford the isolation of the title compound. ¹HNMR (400 MHz, CD₃OD) ppm: 8.32 (br. s., 1H), 7.58-7.66 (m, 2H), 7.46 (d,1H, J=8.1 Hz), 7.14-7.22 (m, 1H), 7.00-7.12 (m, 2H), 6.73 (s, 1H), 4.26(d, 2H, J=12.9 Hz), 3.71 (s, 3H), 3.33-3.37 (m, 1H), 2.9-3.17 (m, 2H),1.85-2.06 (m, 4H); MS (ES+): m/z 391.06 [MH+].

Example 53

3-[1-(4-Chloro-2-methylbenzoyl)piperidin-4-yl]-1-(1H-indol-2-yl)imidazo[1,5-a]pyrazin-8-amine

Prepared according to the procedure described above for EXAMPLE 26,except using 4-chloro-2-methylbenzoic acid in place of formic acid. MS(ES+): m/z 485.05 [MH+].

Example 54

1-(1H-Indol-2-yl)-3-(1-{[1-(4-methylphenyl)cyclopropyl]carbonyl}piperidin-4-yl)imidazo[1,5-a]pyrazin-8-amine

Prepared according to the procedure described above for EXAMPLE 26,except using 1-(4-methylphenyl)cyclopropanecarboxylic acid in place offormic acid. MS (ES+): m/z 491.11 [MH+].

Example 55

3-[1-(4-Chloro-3-methoxybenzoyl)piperidin-4-yl]-1-(1H-indol-2-yl)imidazo[1,5-a]pyrazin-8-amine

Prepared according to the procedure described above for EXAMPLE 26,except using 4-chloro-3-methoxybenzoic acid in place of formic acid. MS(ES+): m/z 501.04 [MH+].

Example 56

1-(5-{[4-(8-Amino-1-(1H-indol-2-yl)imidazo[1,5-a]pyrazin-3-yl)piperidin-1-yl]carbonyl}-2-thienyl)ethanone

Prepared according to the procedure described above for EXAMPLE 26,except using 5-acetylthiophene-2-carboxylic acid in place of formicacid. MS (ES+): m/z 485.04 [MH+].

Example 57

1-(1H-Indol-2-yl)-3-[1-(3-thienylcarbonyl)piperidin-4-yl]imidazo[1,5-a]pyrazin-8-amine

Prepared according to the procedure described above for EXAMPLE 26,except using thiophene-3-carboxylic acid in place of formic acid. MS(ES+): m/z 443.04 [MH+].

Example 58

1-(1H-Indol-2-yl)-3-[1-(4-nitrobenzoyl)piperidin-4-yl]-imidazo[1,5-a]pyrazin-8-amine

Prepared according to the procedure described above for EXAMPLE 26,except using 4-nitrobenzoic acid in place of formic acid. MS (ES+): m/z482.07 [MH+].

Example 59

3-[1-(Butylsulfonyl)piperidin-4-yl]-1-(1H-indol-2-yl)imidazo[1,5-a]pyrazin-8-amine

A solution of1-(1H-indol-2-yl)-3-piperidin-4-ylimidazo[1,5-a]pyrazin-8-aminehydrochloride (33.23 mg, 0.075 mmol) in DMF (1 mL) was treated withN,N-diisopropylethylamine (0.05 mL, 0.3 mmol) and a solution of^(n)butanesulfonyl chloride (9.42 mg, 0.0602 mmol) in 1 mL of DMF. Themixture was left to stir at rt for 1 h and then subjected tomass-directed preparative HPLC to afford the title compound. ¹H NMR (400MHz-DMSO-d6) δ 0.91 (t, 3H), 1.40-1.45 (m, 2H), 1.66-1.69 (m, 2H),1.86-1.90 (m, 2H) 2.04-2.09 (m, 2H) 3.02-3.11 (m, 5H) 3.73-3.77 (m, 2H),6.47 (bs, 2H), 6.64 (s, 1H), 7.00-7.05 (m, 1H) 7.09-7.12 (m, 2H), 7.45(d, J=8.4 Hz, 1H), 7.58 (d, J=8.0 Hz, 1H), 7.69 (d, J=5.2 Hz, 1H). MS(ES+): m/z: 453.24 [MH+].

Example 60

1-(1H-Indol-2-yl)-3-[1-(isopropylsulfonyl)piperidin-4-yl]imidazo[1,5-a]pyrazin-8-amine

Prepared according to the procedure described above for EXAMPLE 59,except using isopropane-2-sulfonyl chloride in place of^(n)butanesulfonyl chloride. MS (ES+): m/z 439.27 [MH+].

Example 61

3-{1-[(4-Fluorophenyl)sulfonyl]piperidin-4-yl}-1-(1H-indol-2-yl)imidazo[1,5-a]pyrazin-8-amine

Prepared according to the procedure described above for EXAMPLE 59,except using 4-fluorobenzenesulfonyl chloride in place of^(n)butanesulfonyl chloride. MS (ES+): m/z 491.15 [MH+].

Example 62

3-{1-[(2,5-Dimethoxyphenyl)sulfonyl]piperidin-4-yl}-1-(1H-indol-2-yl)imidazo[1,5-a]pyrazin-8-amine

Prepared according to the procedure described above for EXAMPLE 59,except using 2,5-dimethoxybenzenesulfonyl chloride in place of^(n)butanesulfonyl chloride. MS (ES+): m/z 533.17 [MH+].

Example 63

1-(1H-Indol-2-yl)-3-{1-[(4-methylphenyl)sulfonyl]piperidin-4-yl}imidazo[1,5-a]pyrazin-8-amine

Prepared according to the procedure described above for EXAMPLE 59,except using 4-methylbenzenesulfonyl chloride in place of^(n)butanesulfonyl chloride. MS (ES+): m/z 487.94 [MH+].

Example 64

3-{1-[(3-Fluorophenyl)sulfonyl]piperidin-4-yl}-1-(1H-indol-2-yl)imidazo[1,5-a]pyrazin-8-amine

Prepared according to the procedure described above for EXAMPLE 59,except using 3-fluorobenzenesulfonyl chloride in place of^(n)butanesulfonyl chloride. MS (ES+): m/z 491.92 [MH+].

Example 65

3-Cyclobutyl-1-(1H-pyrrolo[2,3-b]pyridin-2-yl)imidazo[1,5-a]pyrazin-8-amine

3-Cyclobutyl-1-[1-(2-trimethylsilylethoxymethyl)-1H-pyrrolo[2,3-b]pyridin-2-yl]imidazo[1,5-a]pyrazin-8-amine(35 mg, 0.08 mmol) was stirred with concentrated HCl for 15 min. Themixture was then concentrated in vacuo and purified via mass directedpreparative HPLC to afford the title compound. ¹H NMR (400 MHz DMSO-d6)δ 1.92-2.00 (m, 1H), 2.07-2.14 (m, 1H), 2.43-2.47 (m, 4H), 3.93-4.01 (m,1H), 6.35-6.49 (bs, 2H), 6.64-6.70 (m, 1H), 7.03-7.10 (m, 2H), 7.39-7.49(m, 1H), 7.95-8.00 (m, 1H), 8.18-8.23 (m, 1H), 11.91 (bs, 1H). MS (ES+):m/z: 305.17 [MH+].

Example 66

Methyltrans-4-(8-amino-1-(1H-indol-2-yl)imidazo[1,5-a]pyrazin-3-yl)cyclohexanecarboxylate

Starting from trans-methyl4-(8-chloroimidazo[1,5-a]pyrazin-3-yl)cyclohexanecarboxylate, the titlecompound was prepared according to procedures analogous to thosedescribed for EXAMPLE 10. ¹H NMR (d₆-DMSO, 400 MHz): δ 11.42 (br s, 1H),7.70 (d, J=4.0 Hz, 1H), 7.58 (d, J=8.0 Hz, 1H), 7.46 (d, J=8.0 Hz, 1H),7.30-6.90 (m, 3H), 6.63 (br s, 1H), 6.44 (br s, 1H), 3.64 (s, 3H), 3.18(m, 1H), 2.44 (m, 1H), 2.03 (m, 4H), 1.80-1.50 (m, 4H). MS (ES+): m/z390.28 [MH+].

Example 67

trans-4-(8-Amino-1-(1H-indol-2-yl)imidazo[1,5-a]pyrazin-3-yl)cyclohexanecarboxylicacid

A mixture of 37% HCl (30 mL) and methyltrans-4-(8-amino-1-(1H-indol-2-yl)imidazo[1,5-a]pyrazin-3-yl)cyclohexanecarboxylate(500.0 mg, 1.28 mmol) was stirred for 18 h at rt. The reaction mixturewas then concentrated in vacuo, and the residue washed with diethylether (3×10 mL) and ethyl acetate (2×10 mL), then with ice-coldacetonitrile (10 mL) to afford 0.3 g of the desired product. ¹H NMR(d₆-DMSO, 400 MHz): δ 12.15 (br s, 1H), 11.69 (s, 1H), 8.45 (br s, 2H),7.97 (d, J=6.4 Hz, 1H), 7.63 (d, J=8.0 Hz, 1H), 7.50 (dd, J=8.0, 0.4 Hz,1H), 7.19 (m, 1H), 7.13 (d, J=6.0 Hz, 1H), 7.06 (m, 1H), 6.83 (d, J=1.6Hz, 1H), 3.27 (td, J=11.6, 3.2, 3.2 Hz, 1H), 2.33 (td, J=10.8, 3.2, 3.2Hz, 1H), 2.05 (m, 4H), 1.73 (m, 2H) and 1.58 (m/z, 2H). MS (ES+): m/z376.05 [MH+].

Example 68

trans-4-(8-Amino-1-(1H-indol-2-yl)imidazo[1,5-a]pyrazin-3-yl)-N-pyridin-3-ylcyclohexanecarboxamide

A suspension of 3-aminopyridine (40 mg, 0.43 mmol) in toluene (1.3 mL)was treated with a 2M toluene solution of trimethylaluminum (0.3 mL,0.60 mmol).

After 25 min, the resulting solution was treated with methyltrans-4-(8-amino-1-(1H-indol-2-yl)imidazo[1,5-a]pyrazin-3-yl)cyclohexanecarboxylate(30 mg, 0.08 mol) and the mixture stirred at rt overnight. The mixturewas then stirred with 2M NaOH (20 mL) and ethyl acetate (20 mL) for 10min., then the organic phase was separated and the aqueous extractedEtOAc (3×15 mL). The combined organic extracts were washed with water(20 mL) and brine (20 mL), then dried over Na₂SO₄ and concentrated invacuo to give crude product which was subjected to mass-directedpreparative HPLC to afford pure desired product. ¹H NMR (d₆-DMSO, 400MHz): δ 11.45 (br s, 1H), 10.12 (s, 1H), 8.77 (d, J=2.4 Hz, 1H), 8.25(d, J=4.8 Hz, 1H), 8.14 (s, 1H), 8.08 (dd, J=8.0, 1.6 Hz, 1H), 7.71 (d,J=5.2 Hz, 1H), 7.59 (d, J=7.6 Hz, 1H), 7.46 (d, J=8.4 Hz, 1H), 7.34 (m,1H), 7.15-7.00 (m, 3H), 6.65 (s, 1H), 6.42 (br s, 2H), 3.22 (m, 1H),2.47 (m, 1H), 2.15-1.95 (m, 4H), and 1.85-1.65 (m, 4H). MS (ES+): m/z452.17 [MH+].

Example 69

trans-4-(8-Amino-1-(1H-indol-2-yl)imidazo[1,5-a]pyrazin-3-yl)-N-pyridin-2-ylcyclohexanecarboxamide

Prepared according to the procedure described above for EXAMPLE 68,except using 2-aminopyridine in place of 3-aminopyridine. MS (ES+): m/z452.17 [MH+].

Example 70

trans-4-(8-Amino-1-(1H-indol-2-yl)imidazo[1,5-a]pyrazin-3-yl)-N-phenylcyclohexanecarboxamide

Prepared according to the procedure described above for EXAMPLE 68,except using aniline in place of 3-aminopyridine. MS (ES+): m/z 451.16[MH+].

Example 71

trans-4-[8-Amino-1-(1H-indol-2-yl)imidazo[1,5-a]pyrazin-3-yl]cyclohexanecarboxamide

trans-4-(8-Amino-1-iodoimidazo[1,5-a]pyrazin-3-yl)cyclohexanecarboxamide(40 mg, 0.10 mmol), 1-(tert-butoxycarbonyl)-1H-indole-2-boronic acid (33mg, 0.12 mmol), and sodium carbonate (33 mg, 0.3 mmol) were added toDME:Water (5:1) (2 mL) and the mixture degassed with Argon for 10 min.Tetrakis(triphenylphosphine)palladium(0) (8.0 mg, 0.007 mmol) was thenadded and the reaction mixture microwaved at 110° C. for 1 h, Themixture was concentrated in vacuo, taken up in DMSO, and purified bymass-directed preparative HPLC to afford desired product. ¹H NMR(d₆-DMSO, 400 MHz): □ 11.50 (br s, 1H), 7.72 (m, 1H), 7.58 (m, 1H), 7.46(dd, J=7.6, 0.4 Hz, 1H), 7.25 (br s, 1H), 7.13 (m, 1H), 7.08-7.00 (m,2H), 6.70 (br s, 1H), 6.69 (br s, 1H), 3.16 (m, 1H), 2.20 (m, 1H),2.10-1.80 (m, 4H) and 1.65 (m, 4H). MS (ES+): m/z 375.17 [MH+].

Example 72

trans-4-(8-Amino-1-(1H-indol-2-yl)imidazo[1,5-a]pyrazin-3-yl)-N-ethylcyclohexanecarboxamide

Ethylamine hydrochloride (30 mg, 0.37 mmol),2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate(35 mg, 0.1 mmol), and N,N-diisopropylethylamine (80 μL, 0.53 mmol) wereadded to a solution oftrans-4-(8-Amino-1-(1H-indol-2-yl)imidazo[1,5-a]pyrazin-3-yl)cyclohexanecarboxylicacid (25 mg, 0.07 mmol) in anhydrous DMF (2 mL). Upon completion ofreaction (as monitored by LCMS), the mixture was added to a saturatedaqueous sodium bicarbonate solution (10 mL). The resulting precipitatewas collected by filtration and washed with cold acetonitrile (3×10 mL)to afford 13 mg of the desired product. ¹H NMR (d₆-DMSO, 400 MHz): δ11.41 (br s, 1H), 7.75 (dd, J=4.0, 4.0 Hz, 1H), 7.69 (d, J=4.0 Hz, 1H),7.58 (d, J=8.0, 4.0 Hz, 1H), 7.45 (d, J=4.0, 4.0 Hz, 1H), 7.12 (dd,J=8.0, 8.0 Hz, 1H), 7.08-7.00 (m, 2H), 6.63 (m, 1H), 6.43 (br s, 2H),3.16 (m, 1H), 3.07 (m, 2H), 2.18 (m, 1H), 2.02 (m, 2H), 1.84 (m, 2H),1.66 (m, 4H) and 1.02 (t, J=4.0 Hz, 3H). MS (ES+): m/z 403.09 [MH+].

Example 73

trans-4-(8-Amino-1-(1H-indol-2-yl)imidazo[1,5-a]pyrazin-3-yl)-N-cyclopropylcyclohexanecarboxamide

Prepared according to the procedure described above for EXAMPLE 72,except using cyclopropylamine in place of ethylamine. MS (ES+): m/z415.22 [MH+].

Example 74

Benzyl{[trans-4-(8-amino-1-(1H-indol-2-yl)imidazo[1,5-a]pyrazin-3-yl)cyclohexyl]methyl}carbamate

A mixture of benzyl{[trans-4-(8-amino-1-iodoimidazo[1,5-a]pyrazin-3-yl)cyclohexyl]methyl}carbamate(1.00 g, 0.00180 mol), 1-(tert-butoxycarbonyl)-1H-indole-2-boronic acid(0.517 g, 0.00198 mol), 1,2-dimethoxyethane (7.7 mL), water (1.4 mL,0.081 mol) and Cesium Carbonate (1.17 g, 0.00360 mol) degassed threetimes, treated with tetrakis(triphenylphosphine)palladium(0) (200 mg,0.0002 mol) and degassed once more. The resulting mixture was heated at100° C. overnight before being diluted with EtOAc (40 mL), washed withwater (2×30 mL) and brine (20 mL) then dried over Na₂SO₄ andconcentrated in vacuo. The crude product thus isolated waschromatographed over silica gel eluting with hexane→EtOAc:hexane: 5% 2MNH₃ in MeOH 1:1:0.05 to afford the title compound. ¹H NMR (400 MHz,CDCl₃): δ 1.13-1.22 (m, 2H), 1.75-1.86 (m, 2H), 1.94-1.97 (m, 2H),2.11-2.13 (m, 2H), 2.86 (m, 1H), 3.12-3.16 (m, 2H), 4.82 (m, 1H), 5.12(s, 2H), 5.69 (br, 2H), 6.78 (s, 1H), 7.13-7.15 (m, 2H), 7.19-7.25 (m,2H), 7.32-7.38 (m, 5H), 7.42 (d, J=8.0 Hz, 1H), 7.64 (d, J=8.4 Hz, 1H),9.09 (br, 1H). MS (ES+): m/z 495 [MH+].

Example 75

N-{[trans-4-(8-Amino-1-(1H-indol-2-yl)imidazo[1,5-a]pyrazin-3-yl)cyclohexyl]methyl}-3-furamide

Prepared according to the procedure described above for EXAMPLE 22,except using 2-furoic acid in place of acetic acid. MS (ES+): m/z 455.20[MH+].

Example 76

N-{[trans-4-(8-Amino-1-(1H-indol-2-yl)imidazo[1,5-a]pyrazin-3-yl)cyclohexyl]methyl}benzamide

Prepared according to the procedure described above for EXAMPLE 22,except using benzoic acid in place of acetic acid. MS (ES+): m/z 465.25[MH+].

Example 77

N-{[trans-4-(8-Amino-1-(1H-indol-2-yl)imidazo[1,5-a]pyrazin-3-yl)cyclohexyl]methyl}cyclobutanecarboxamide

Prepared according to the procedure described above for EXAMPLE 22,except using cyclobutanecarboxylic acid in place of acetic acid. MS(ES+): m/z 443.25 [MH+].

Example 78

N-{[trans-4-(8-Amino-1-(1H-indol-2-yl)imidazo[1,5-a]pyrazin-3-yl)cyclohexyl]methyl}-3,5-dimethoxybenzamide

Prepared according to the procedure described above for EXAMPLE 22,except using 3,5-dimethoxybenzoic acid in place of acetic acid. MS(ES+): m/z 525.35 [MH+].

Example 79

N-{[trans-4-(8-Amino-1-(1H-indol-2-yl)imidazo[1,5-a]pyrazin-3-yl)cyclohexyl]methyl}-2,4-dimethoxybenzamide

Prepared according to the procedure described above for EXAMPLE 22,except using 2,4-dimethoxybenzoic acid in place of acetic acid. MS(ES+): m/z 525.33 [MH+].

Example 80

N-{[trans-4-(8-Amino-1-(1H-indol-2-yl)imidazo[1,5-a]pyrazin-3-yl)cyclohexyl]methyl}formamide

Prepared according to the procedure described above for EXAMPLE 22,except using formic acid in place of acetic acid. MS (ES+): m/z 389.10[MH+].

Example 81

(1R,2R)—N-{[trans-4-(8-amino-1-(1H-indol-2-yl)imidazo[1,5-a]pyrazin-3-yl)cyclohexyl]methyl}-2-phenylcyclopropanecarboxamide

Prepared according to the procedure described above for EXAMPLE 22,except using (1R,2R)-2-phenylcyclopropanecarboxylic acid in place ofacetic acid. MS (ES+): m/z 505.30 [MH+].

Example 82

N-{[trans-4-(8-Amino-1-(1H-indol-2-yl)imidazo[1,5-a]pyrazin-3-yl)cyclohexyl]methyl}-3-chloro-6-fluorobenzo[b]thiophene-2-carboxamide

Prepared according to the procedure described above for EXAMPLE 22,except using 3-chloro-6-fluorobenzo[b]thiophene-2-carboxylic acid inplace of acetic acid. MS (ES+): m/z 573.35 & 575.31 [MH+].

Example 83

N-{[trans-4-(8-Amino-1-(1H-indol-2-yl)imidazo[1,5-a]pyrazin-3-yl)cyclohexyl]methyl}isoquinoline-2-carboxamide

Prepared according to the procedure described above for EXAMPLE 22,except using isoquinoline-2-carboxylic acid in place of acetic acid. MS(ES+): m/z 516.40 [MH+].

Example 84

N-{[trans-4-(8-Amino-1-(1H-indol-2-yl)imidazo[1,5-a]pyrazin-3-yl)cyclohexyl]methyl}indole-3-carboxamide

Prepared according to the procedure described above for EXAMPLE 22,except using indole-3-carboxylic acid in place of acetic acid. MS (ES+):m/z 505.46 [MH+].

Example 85

1-(4-Chloro-1H-indol-2-yl)-3-cyclobutylimidazo[1,5-a]pyrazin-8-amine

Prepared according to the procedure described above for EXAMPLE 2,except using 1-(tert-butoxycarbonyl)-4-chloro-1H-indole-2-boronic acidin place of 1-(tert-butoxycarbonyl)-1H-indole-2-boronic acid. ¹H NMR(400 MHz-DMSO-d6) δ 1.91-1.98 (m, 1H), 2.08-2.15 (m, 1H), 2.42-2.46 (m,4H), 3.97-4.00 (m, 1H), 6.42 (bs, 2H), 6.67 (s, 1H), 7.09-7.14 (m, 3H),7.43-7.47 (m, 2H) and 11.83 (bs, 1H). MS (ES+): m/z 338.26 [MH+].

Example 86

1-(1H-Indol-2-yl)-3-[1-(4-methoxyphenyl)cyclopropyl]imidazo[1,5-a]pyrazin-8-amine

Prepared according to the procedure described above for EXAMPLE 2,except using 4-methoxyphenylcyclopropanecarboxylic acid in place ofcyclobutanecarboxylic acid. ¹H NMR (400 MHz, CHLOROFORM-d) δ ppm 1.46(s, 2H), 1.58 (s, 2H), 3.76 (s, 3H), 6.78 (d, J=8.80 Hz, 2H), 6.77 (s,1H), 6.82 (s, 1H), 6.98 (d, J=5.13 Hz, 1H), 7.03 (d, J=8.80 Hz, 2H),7.15 (t, J=7.52 Hz, 1H), 7.23 (s, 2 H), 7.44 (d, J=8.07 Hz, 1H), 7.65(d, J=8.07 Hz, 1H) and 9.36 (br. s., 1H). MS (ES+): m/z 396.15 [MH+].

Example 87

1-(1H-Indol-2-yl)-3-[1-(propylsulfonyl)piperidin-4-yl]imidazo[1,5-a]pyrazin-8-amine

Prepared according to the procedure described above for EXAMPLE 59,except using propane-2-sulfonyl chloride in place of ^(n)butanesulfonylchloride. MS (ES+): m/z 439.06 [MH+].

Example 88

1-(1H-Indol-2-yl)-3-[1-(phenylsulfonyl)piperidin-4-yl]imidazo[1,5-a]pyrazin-8-amine

Prepared according to the procedure described above for EXAMPLE 59,except using benzenesulfonyl chloride in place of ^(n)butanesulfonylchloride. MS (ES+): m/z 473.29 [MH+].

Example 89

1-(1H-Indol-2-yl)-3-{1-[(3,3,3-trifluoropropyl)sulfonyl]piperidin-4-yl}imidazo[1,5-a]pyrazin-8-amine

Prepared according to the procedure described above for EXAMPLE 59,except using 3,3,3-trifluoropropane-1-sulfonyl chloride in place of^(n)butanesulfonyl chloride. MS (ES+): m/z 493.19 [MH+].

Example 90

trans-3-(8-Amino-1-(1H-indol-2-yl)imidazo[1,5-a]pyrazin-3-yl)-N-[(1S)-1-phenylethyl]cyclohexanecarboxamide

Prepared according to the procedure described above for EXAMPLE 72,except using (1S)-1-phenylethanamine in place of cyclopropylamine. MS(ES+): m/z 479.11 [MH+].

Example 91

N-{[trans-4-(8-Amino-1-(1H-indol-2-yl)imidazo[1,5-a]pyrazin-3-yl)cyclohexyl]methyl}(3-bromophenyl)acetamide

Prepared according to the procedure described above for EXAMPLE 22,except using 3-bromophenylacetic acid in place of acetic acid. MS (ES+):m/z 557.21 and 559.20 [MH+].

Example 92

N-{[trans-4-(8-Amino-1-(1H-indol-2-yl)imidazo[1,5-a]pyrazin-3-yl)cyclohexyl]methyl}(2,6-dichloro-5-fluoropyridin-3-yl)acetamide

Prepared according to the procedure described above for EXAMPLE 22,except using (2,6-dichloro-5-fluoropyridin-3-yl)acetic acid in place ofacetic acid. MS (ES+): m/z 522.21 [MH+].

Example 93

Benzyl4-[8-amino-1-(1H-indol-5-yl)imidazo[1,5-a]pyrazin-3-yl]piperidine-1-carboxylate

Prepared according to the procedure described above for EXAMPLE 24,except using indole-5-boronic acid in place of1-(tert-butoxycarbonyl)-1H-indole-2-boronic acid. MS (ES+): m/z 494.97[MH+].

Example 94

trans-4-(8-Amino-1-(1H-indol-2-yl)imidazo[1,5-a]pyrazin-3-yl)-N-benzimidazol-2-ylcyclohexanecarboxamide

Prepared according to the procedure described above for EXAMPLE 68,except using 2-aminobenzimidazole in place of 3-aminopyridine. MS (ES+):m/z 490.97 [MH+].

Example 95

1-(1H-Indol-2-yl)-3-[1-(quinolin-2-ylmethyl)piperidin-4-yl]imidazo[1,5-a]pyrazin-8-amine

A solution of1-(1H-Indol-2-yl)-3-piperidin-4-ylimidazo[1,5-a]pyrazin-8-aminehydrochloride (30 mg, 0.09 mmol), 2-formylquinoline (17 mg, 0.11 mmol)and triethylamine (0.019 mL, 0.14 mmol) in 1,4-dioxane (1 mL) wastreated with sodium cyanoborohydride (5.7 mg, 0.090 mmol) and microwavedat 300 watts, 120° C. for 20 min. The mixture was concentrated in vacuo,the residue was dissolved in methanol loaded onto an SCX ion exchangecartridge, and then eluted with 1M NH₄OH in methanol. The semi-purematerial thus obtained was then subjected to semi-preparative HPLC toafford desired product. ¹H NMR (400 MHz, MeOD) δ ppm 2.13-2.33 (m, 4H),2.90 (t, J=10.86, 9.60 Hz, 2H), 3.47 (d, J=10.11 Hz, 2H), 4.29 (s, 2H),6.74 (s, 1H), 7.02-7.11 (m, 2H), 7.19 (t, J=8.08, 7.07 Hz, 1H), 7.47 (d,J=9.09 Hz, 1H), 7.58-7.65 (m, 3H), 7.69 (d, J=8.59 Hz, 1H), 7.80 (t,J=8.34, 6.82 Hz, 1H), 7.96 (d, J=7.33 Hz, 1H), 8.08 (d, J=8.34 Hz, 1H)and 8.39 (d, J=8.59 Hz, 1H). MS (ES+): m/z 474.23 [MH+].

Example 96

1-(1H-Indol-2-yl)-3-[1-(2-thienylsulfonyl)piperidin-4-yl]imidazo[1,5-a]pyrazin-8-amine

Prepared according to the procedure described above for EXAMPLE 59,except using thiophene-2-sulfonyl chloride in place of^(n)butanesulfonyl chloride. MS (ES+): m/z 479.16 [MH+].

Example 97

1-(1H-Indol-2-yl)-3-{1-[(3-methylphenyl)sulfonyl]piperidin-4-yl}imidazo[1,5-a]pyrazin-8-amine

Prepared according to the procedure described above for EXAMPLE 59,except using 3-methylbenzenesulfonyl chloride in place of^(n)butanesulfonyl chloride. MS (ES+): m/z 487.94 [MH+].

Example 98

1-(1H-Indol-2-yl)-3-{1-[(1-methyl-1H-imidazol-4-yl)sulfonyl]piperidin-4-yl}imidazo[1,5-a]pyrazin-8-amine

Prepared according to the procedure described above for EXAMPLE 59,except using 1-methyl-1H-imidazole-4-sulfonyl chloride in place of^(n)butanesulfonyl chloride. MS (ES+): m/z 477.20 [MH+].

The following examples were prepared according to procedures analogousto those described above, utilizing where necessary known literaturechemistries.

Ex Ex # Structure MH+ # Structure MH+  99

500.93 502.91 100

433.06 101

433.02 102

404.96 103

474.23 104

483.00 105

483.27 106

452.04 107

514.92 108

500.89 109

492.92 110

447.01 111

498.93 500.94 112

456.90 113

420.97 114

496.91 115

488.91 116

475.91 117

468.84 118

426.99 119

461.00 120

320.86 121

391.23 122

490.97 123

493.18 124

487.09 125

459.01 126

446.15 127

452.98 128

451.97 129

481.95 130

470.00 131

535.91 132

454.97 133

448.02 134

318.03 135

470.96 136

475.92 137

475.92 138

457.08 139

426.92 140

521.03 523.08 141

427.05 142

457.02 143

444.20 144

425.91 145

376.98 146

337.97 339.92 147

304.95 148

452.95 149

305.20 150

389.83 151

426.97 152

456.79 153

443.97 154

475.94 155

492.76 156

475.85 157

460.13 158

375.98 159

466.97 160

451.98 161

304.19 162

405.02 163

433.18 164

532.90 165

476.95 166

410.02 167

321.92 168

333.87 169

381.83 383.72 170

495.97 171

465.96 232

440.89 442.86 172

468.84 470.50 233

354.74 356.98 173

480.20 234

335.84 174

452.97 235

442.96 175

466.20 236

480.98 176

399.92 237

421.83 177

426.91 238

549.91 178

472.62 239

482.02 179

550.68 552.50 240

419.89 180

456.63 241

467.92 181

471.89 242

420.97 182

523.93 243

487.97 183

496.05 244

319.00 184

510.00 245

405.03 185

483.89 246

499.95 501.96 186

404.18 247

423.83 425.93 187

427.93 248

409.95 411.90 188

428.88 249

376.99 189

460.66 250

412.06 414.03 191

548.72 251

404.96 192

456.86 252

391.01 193

525.25 253

419.12 194

467.21 254

434.04 195

486.96 255

405.03 196

470.97 256

445.01 197

444.00 257

438.94 440.89 198

363.89 258

406.98 406.99 199

403.07 259

421.00 200

458.97 260

437.93 439.95 201

500.96 261

511.21 513.14 202

500.94 262

447.03 203

362.03 263

461.05 204

473.95 264

447.99 205

335.06 265

462.00 206

433.07 266

387.20 207

486.96 267

432.06 208

348.02 268

434.02 434.06 209

473.87 269

437.97 439.95 210

334.88 270

468.95 211

457.95 271

423.97 425.93 212

318.92 272

448.05 213

475.02 273

471.98 214

425.17 427.06 274

418.09 215

518.83 275

405.03 216

379.87 276

363.98 217

439.19 277

423.97 425.99 218

526.77 278

391.01 219

483.04 279

460.94 220

520.89 280

421.00 221

436.98 438.94 281

435.03 222

425.93 282

485.32 223

411.13 413.02 283

510.38 224

453.99 284

406.29 225

433.02 285

404.21 226

479.85 286

420.53 227

434.95 287

417.29 228

417.21 288

423.29 229

500.99 502.88 289

455.11 457.09 230

516.91 518.90 290

497.93 231

498.02 291

424.04 425.99 292

434.08 333

406.35 406.42 293

475.89 334

501.31 294

461.94 335

487.44 295

485.14 487.10 336

420.15 420.18 296

491.18 337

411.06 413.07 297

488.63 338

471.35 298

434.08 339

454.07 456.03 299

435.10 340

484.44 300

505.10 341

470.41 301

438.00 342

469.46 302

432.02 343

409.35 303

467.30 344

416.17 304

455.23 345

485.39 305

405.09 346

444.10 306

424.13 426.23 347

471.41 307

458.99 348

404.04 406.06 308

409.97 411.96 349

404.27 406.29 309

445 445.1 350

469.39 310

407.05 351

401.39 311

421.00 352

444.16 312

512.40 353

481.12 483.14 313

418.03 354

415.17 314

391.06 355

400.09 315

453.04 453.17 453.39 356

425.34 427.33 316

474.95 357

417.36 317

457.08 358

402.33 318

457.95 359

381.96 384.01 319

482.96 360

487.01 489.03 320

483.90 361

495.03 321

390.02 362

428.02 322

463.08 363

458.32 323

460.09 364

487.01 488.90 324

480.21 365

470.37 325

471.11 366

521.27 523.27 326

455.94 367

443.22 327

486.20 368

459.28 328

436.23 438.26 369

458.37 329

432.02 370

493.22 495.18 330

402.06 371

471.03 331

452.12 372

510.03 332

434.25 373

496.06 374

501.45 375

493.49 376

507.46 377

569.56 378

507.46 379

521.50 380

583.53 381

425.39 382

439.42 383

555.55 384

569.55

The following compounds are expected to be active as inhibitors of mTOR.Where shown, X can be N or CH.

Cell lines: Human cancer cell lines were purchased from the AmericanType Culture Collection (ATCC). The cell lines H460, Calu6, SW1573,H1703, H292, H358, HCT-116, HT-29, FET, GEO, SW480, Colo205 CBS, BxPC3,HPAC, CFPAC, MiaPaca-2, Panc1, MDA-MB-468, BT-20, MDA-MB-435 H441, H322,A1165, Igrov-1, Ovcar-3, CA-OV-3, MDAH2774, SW626, SKOV-3, Cal-27, RPMI2650, and MDA-MB-231 were grown in media as prescribed by the ATCC,containing 10% FCS. Hsc-2, Hsc-4 and OVK-18 were obtained from the RikenCell Bank and were cultured according to the Riken Cell Bank recommendedconditions. HNSCC 1483, HNSCC 1386, HNSCC 1186 were a gift from MemorialSloan Kettering and were cultured in 1:1 DMEM:Hams F12 with 10% FCS.Ovcar-4, Ovcar-5 and Ovcar-8 were obtained from the NCI and were grownin RPMI with 10% FCS. HN5 was a gift from an academic investigator andwas cultured in DMEM plus 10% FCS.

Measurement of Cell Proliferation: Cell proliferation was determinedusing the Cell Titer Glo assay (Promega Corporation, Madison, Wis.).Cell lines were seeded at a density of 3000 cells per well in a 96-wellplate. 24 hours after plating cells were dosed with varyingconcentrations of drug, either as a single agent or in combination. Thesignal for Cell Titer Glo was determined 72 hours after dosing.

Measurement of apoptosis: Induction of apoptosis as measured byincreased Caspase 3/7 activity was determined using the Caspase 3/7 Gloassay (Promega Corporation, Madison, Wis.). Cell lines were seeded at adensity of 3000 cells per well in a 96-well plate. 24 hours afterplating cells were dosed with varying concentrations of drug, either asa single agent or in combination. The signal for Caspase 3/7 Glo wasdetermined 24 hours after dosing. The caspase 3/7 activity wasnormalized to cell number per well, using a parallel plate treated withCell Titer Glo (Promega Corporation, Madison, Wis.). Signal for eachwell was normalized using the following formula: Caspase 3/7 Gloluminescence units/Cell Titer Glo fraction of DMSO control. All graphswere generated using PRISM® software (Graphpad Software, San Diego,Calif.).

Analysis of Additivity and Synergy: The Bliss additivism model was usedto classify the effect of combining rapamycin and erlotinib as additive,synergistic, or antagonistic. A theoretical curve was calculated forcombined inhibition using the equation:E_(bliss)=E_(A)+E_(B)−E_(A)*E_(B), where E_(A) and E_(B) are thefractional inhibitions obtained by drug A alone and drug B alone atspecific concentrations. Here, E_(bliss) is the fractional inhibitionthat would be expected if the combination of the two drugs was exactlyadditive. If the experimentally measured fractional inhibition is lessthan E_(bliss) the combination was said to be synergistic. If theexperimentally measured fractional inhibition is greater than E_(bliss)the combination was said to be antagonistic. For dose response curves,the Bliss additivity value was calculated for varying doses of drug Awhen combined with a constant dose of drug B. This allowed an assessmentas to whether drug B affected the potency of drug A or shifted itsintrinsic activity. All plots were generated using PRISM® software(Graphpad Software, San Diego, Calif.).

Preparation of Protein Lysates and Western Blotting:

Cell extracts were prepared by detergent lysis (50 mM Tris-HCl, pH 8.0,150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, containingprotease inhibitor (P8340, Sigma, St. Louis, Mo.) and phosphataseinhibitor (P5726, Sigma, St. Louis, Mo.) cocktails. The soluble proteinconcentration was determined by micro-BSA assay (Pierce, Rockford Ill.).Protein immunodetection was performed by electrophoretic transfer ofSDS-PAGE separated proteins to nitrocellulose, incubation with antibody,and chemiluminescent second step detection (PicoWest; Pierce, Rockford,Ill.). The antibodies included: EGFR, phospho-EGFR (Y1068), ErbB2,phospho-erbB2, ErbB3, phospho-ErbB3, ErbB4, phospho-p42/p44,phospho-Akt(473), phospho-Akt(308), total Akt, phosho-S6 (235/236), andtotal S6. With the exception of total ErbB3 (obtained from Santa CruzBiotechnology, Inc., Santa Cruz, Calif.), all antibodies were obtainedfrom Cell Signaling Technology, Inc. (Danvers, Mass.). For analysis oferlotinib's effect on the phosphorylation of downstream signalingproteins, cell lines were grown to approximately 70% confluency, atwhich time erlotinib was added at the indicated concentration, and cellswere incubated at 37° C. for two hours. Where indicated, 10 ng/ml EGFligand was added for 5 minutes. The media was removed, cells were washedtwo times with PBS, and cells were lysed as previously described.

Xenograft Experiments:

Female CD-1 nu/nu mice (Charles River Laboratories, Wilmington, Mass.)were implanted with harvested Calu-6 NSCLC tumor cells in a singlesubcutaneous site on the flank of the mice in the axillary region.Tumors were allowed to grow to 200+50 mm³, at which time the animalswere sorted into treatment groups (see below) of 8 animals per group,having approximately equal body weight (+/−1 g) within a group, andtattooed on the tail for permanent identification. Tumor volumes andbody weights were determined twice weekly. The tumor volume wasdetermined by measuring in two directions with vernier calipers andcalculated using the formula: Tumor volume=(length×width²)/2. The datawere plotted as the % change in mean values of tumor volume and bodyweight for each group. The tumor growth inhibition (% TGI) wasdetermined as % TGI=100(1−W_(t)/W_(c)): where Wt is the median tumorvolume of the treated group at time x and We is the median tumor volumeof the control group at time x. Tarceva was dosed in a 6% Captisol(CyDex, Inc) in WFI (Water for Injection) solution and all controlanimals were dosed with an equal volume of the vehicle. Tarceva animalswere dosed by oral gavage once a day for 18 days and the % TGI measuredon day 19. Rapamycin was dosed in 4% ethanol/5% PEG400/5% Tween 80 inWFI. Rapamycin animals were dosed by intraperitoneal injection on day 1,8 and 15, and all control animals were dosed with an equal volume of thevehicle.

Treatment groups:

set 1=vehicle control.

set 2=Tarceva® alone 100 mg/kg daily.

set 3=rapamycin alone 4 mg/kg (day 1/8/15).

set 4=Tarceva®+Rapamycin (4 mg/kg)−(day 1/8/15).

Results

Studies on the effect of a combination of an EGFR kinase inhibitor andan mTOR inhibitor on NSCLC, colon, pancreatic, and breast tumor cells.

The sensitivities to erlotinib of 22 cell lines derived from NSCLC,colon, pancreatic, and breast tumors was determined. The ability of 10μM erlotinib to inhibit the growth of these cell lines is shown inFIG. 1. It was found that these cell lines display a range ofsensitivities to erlotinib, and a maximal growth inhibition of greaterthan 50% was chosen as a cutoff criteria for indicating highsensitivity. Erlotinib's effects on the PI3K-Akt-mTOR pathway in thisgroup of cell lines was analyzed. The three most sensitive cell lines(H292, H358, and BxPC3) and three relatively insensitive cell lines(H460, Calu6, and HCT-116) were selected for further investigation.Xenografts experiments have previously confirmed our in vitroclassification of sensitivity for this group of cell lines (data notshown, and Thompson, S. et al. (2005) Cancer Res. 65(20):9455-9462, forH460 and Calu6). Phosphorylation of S6 was chosen as a readout for theactivity of mTOR. In the three sensitive epithelial cell lines it wasfound that erlotinib can efficiently down-regulate the phosphorylationof pS6, while in the relatively insensitive cell lines, which haveundergone EMT, a less pronounced down-regulation of S6 phosphorylationwas observed. These results are consistent with previous reportsdescribing signaling pathways in other cell lines that are sensitive orrelatively insensitive to EGFR inhibition (Engelman, J. A. et al. (2005)Proc. Natl. Acad. Sci. U.S.A. 102:3788-3793; Moasser, M. M. et al.(2001) Cancer Res. 61:7184-718).

Previous studies with glioblastoma and renal cell carcinoma havedemonstrated the potential of combining EGFR inhibitors with rapamycinfor these two cancer types (Engelman, J. A. et al. (2005) Proc. Natl.Acad. Sci. U.S.A. 102:3788-3793; Moasser, M. M. et al. (2001) CancerRes. 61:7184-718; Gemmill, R. M. et al. (2005) Br. J. Cancer92:2266-2277; Goudar, R. K. et al. (2005) Mol. Cancer Ther. 4:101-112).For example, renal cell carcinomas harboring a mutation in the vonHippel-Lindau gene (VHL) or those with tuberous sclerosis (TSC)mutations were sensitized to the combination of gefitinib and rapamycin(Gemmill, R. M. et al. (2005) Br. J. Cancer 92:2266-2277). Thecombination of EGFR inhibitors with inhibitors of mTOR has also shownpromise in glioblastoma, a tumor type that is characterized by a veryhigh frequency of truncating mutations for the EGFR (EGFRvIII). Thepresent inventors sought to determine if other tumor types that are notcharacterized by these groups of specific mutations might also respondto the combination of EGFR inhibitors with mTOR inhibitors. Cell linesderived from colon, lung, breast, and pancreatic cancers were chosen forfurther analysis. The effect of low doses of rapamycin alone on thegrowth of the cell line panel was initially tested. FIG. 3A shows themaximal inhibition of the cell lines to rapamycin. Several cell lines,including H292 and BxPC3, show some growth inhibition by rapamycinalone. In all cell lines it was verified that rapamycin couldeffectively down-regulate the phosphorylation of S6. A representativepanel is shown in FIG. 4. These results demonstrate how inhibition ofthe mTOR pathway alone might not be sufficient to substantially affectcell proliferation.

The effect that combining rapamycin with erlotinib had on twocriteria: 1. potency and 2. intrinsic efficacy was determined. Ourassessment of additivity, synergy, or antagonism was based on the Blissadditivism model described in the materials and methods section. Thismodel was chosen over isobologram or combination index (CI) analyses asit would allow the evaluation of the nature of drug interactions even incases where the maximal effects for rapamycin or erlotinib as singleagents were low enough such that a reliable IC₅₀ value could not beobtained. For cell lines that are relatively insensitive to erlotinib asa single agent, the IC₅₀ value is often greater than 10 μM, thesolubility limit for the drug. In addition, as both isobologram andcombination index analyses are based upon a single criteria, IC₅₀ value,and do not directly reflect variability within data, they do notnecessarily always indicate whether the extent of synergy issignificant. The Bliss additivism model also allows us to evaluatechanges in intrinsic efficacy. Both isobologram and CI analyses reflectonly changes in potency, but shifts in intrinsic efficacy also have thepotential to be clinically meaningful. This approach has been describedpreviously for high throughput studies involving drug combinations(Borisy, A. A. et al. (2003) Proc. Natl. Acad. Sci. U.S.A.100:7977-7982).

The data for combination of erlotinib with rapamycin are summarized inFIG. 3B. The fractional increase in maximal intrinsic efficacy whenerlotinib is combined with rapamycin is shown (FIG. 3B). The data areexpressed as percentage decrease in cell growth above what would beexpected if the combination was strictly additive in nature. HereBliss=0 would indicate the combination was directly additive, Bliss>0would indicate the percentage increase in maximal inhibition aboveadditivity (synergy), and Bliss<0 would indicate the percentage decreasein maximal inhibition from additivity (antagonism). Synergy, as noted bya positive Bliss value, was observed in 13 of 22 cell lines. Six of thenine erlotinib-sensitive cell lines showed pure additivity (Bliss=0,approximately) with rapamycin, the exceptions being HCT15, MDA-MB-468and HPAC, which showed synergy. The three cell lines that are mostsensitive to erlotinib (i.e. H292, H358, and BxPC3) showed pureadditivity. However, these two targeted agents act synergistically toinhibit cell growth in 10 out of 13 of the cell lines that arerelatively insensitive to erlotinib. In no cell line tested was thecombination of erlotinib and rapamycin antagonistic. HCT-116 and MDA-468were among those that showed synergy. Both of these cell lines have beenreported to contain genetic defects that result in activation of mTOR inan EGFR-independent manner. HCT-116 has a mutation in PI3K, leading toconstitutive activity that is growth factor independent. MDA-468 hasbeen reported to have a deletion of PTEN. The loss of this PI3K pathwayinhibitor leads to sustained activation of mTOR that is partiallyindependent of EGFR. By targeting the Akt-mTOR pathway downstream ofthese mutations one can effectively shut it down. Indeed, rapamycin canfully shut down this pathway as shown by its ability to fully block S6phosphorylation. The sum of inhibition is greater than the effect ofeach targeted agent alone.

The effect of varying concentrations of rapamycin on growth inhibitionin the presence and absence of 10 μM erlotinib is shown in FIG. 5. HereBliss represents the theoretical curve that should be expected if thecombination of erlotinib and rapamycin was purely additive in nature.For the three most sensitive cell lines (H292, H358, and BxPC3) Blissanalysis shows that erlotinib is exactly additive with rapamycin. Thiswas confirmed by isobologram analysis of the data (not shown). For thethree cell lines selected above that are relatively insensitive toerlotinib (H460, Calu6, and HCT-116) it was found that the combinationshows synergy. This is reflected by both an increase in potency and again in maximal intrinsic efficacy. For example, H460 cells show anincrease in maximal efficacy of approximately 60% (34% to 56%) as wellas a 10-fold shift in potency (267 μM to 21 μM) when rapamycin iscombined with erlotinib.

To investigate whether the above in vitro observations would extend toxenograft models in vivo, the combination of erlotinib and rapamycin wastested in a Calu6 xenograft model. As predicted by the cell culturestudies, neither erlotinib or rapamycin had a substantial effect oninhibiting tumor growth as a single agent. However, when these twotargeted agents were combined they showed better than additive effectsin inhibiting tumor growth. These results are shown in FIG. 6. On day 19there is a 56% reduction in tumor growth with the erlotinib andrapamycin combination while there is no statistically significantreduction in tumor growth by either drug when they are administered assingle agents. By day 19 tumors in control animals as well as tumors inanimals treated with either erlotinib or rapamycin alone had grown tothe point that animals had to be sacrificed while animals receiving thecombination continued to survive for greater than 29 days (data notshown).

Discussion:

Molecularly targeted agents acting on EGFR down modulate a number ofdifferent signaling cascades within the cell. The down-regulation of anysingle pathway may be necessary but not sufficient to inhibit tumor cellgrowth. For EGFR inhibitors down-regulation of the PI3K-Akt-mTOR pathwayappears to track with sensitivity to growth inhibition. In cell linesthat are relatively insensitive to blockade of the EGFR, the mTORpathway appears to be insufficiently inhibited. Mechanisms potentiallyleading to the sustained activity of the mTOR pathway include: othergrowth factor receptors including IGF1R and FGFR, specific mutationssuch as constitutively activating PI3K mutations, or loss of PTENactivity. Therefore, in cell lines that are relatively insensitive toEGFR kinase inhibitor, intervention at multiple points may be necessary.The effects of combining the small molecule EGFR kinase inhibitorerlotinib with the mTOR inhibitor rapamycin were determined. It wasfound that cell lines that are relatively insensitive to erlotinib aresensitized to erlotinib by treatment with rapamycin. This is reflectedin both a shift in potency and increase in maximal intrinsic activity.

The rapamycin and erlotinib combination was further characterized in aCalu6 xenograft model. Neither rapamycin or erlotinib showed significantreduction in tumor growth as single agents but show pronounced reductionin tumor growth in combination.

The above results provide an improved understanding of the mechanism ofaction of EGFR inhibitors to inhibitor tumor growth and will assistphysicians in selecting patient populations who are most likely tobenefit from combined therapy compared to single agent therapy.

In summary, in this study it was demonstrated that a combination of anEGFR kinase inhibitor and an mTOR inhibitor can have a synergistic orsupra-additive inhibitory effect on the growth of human breast, colon,NSCL or pancreatic cancer cells. Thus mTOR inhibitors can act as agentsthat can sensitize tumor cells to the effects of EGFR kinase inhibitors.

Studies on the effects on tumor cells of a combination of an EGFR kinaseinhibitor and an mTOR inhibitor that binds to and directly inhibits bothmTORC1 and mTORC2 kinases.

The sensitivities to Compound A of 23 cell lines derived from ovarian,NSCLC, pancreatic, and HNSCC tumors was determined. The ability of 20 μMCompound A to inhibit the growth of these cell lines is shown in FIG. 7.A maximal growth inhibition of greater than 50% was chosen as thecriteria for high sensitivity and all but one of these can becategorized as very sensitive. When combined with erlotinib, Compound Asynergistically inhibits cellular proliferation in the majority of celllines tested. These findings are summarized in FIG. 8 (NSCLC andpancreatic) and FIG. 9 (ovarian, HNSCC and breast). In every cell linetested, the combination improved maximal efficacy and, wheresynergistic, the combination resulted in a reduced EC50. Synergy wasassessed using the bliss additivity model as previously described. In apanel of NSCLC and pancreatic cell lines, the combination of compound Aand erlotinib was synergistic in the four mesenchymal cell lines andadditive in the four epithelial cell lines tested. The effect of varyingconcentrations of Compound A on growth inhibition in the presence andabsence of erlotinib is shown in FIG. 10 (NSCLC) and FIG. 11(pancreatic). Here Bliss represents the theoretical curve that would bepredicted if the combination of erlotinib and Compound A was purelyadditive. For the four mesenchymal NSCLC cell lines (FIG. 10A) Blissanalysis indicates that the combination of erlotinib and Compound A issynergistic. This is reflected by both an increase in potency and a gainin maximal intrinsic efficacy. For the four epithelial NSCLC cell lines(FIG. 10B), the combination is additive. In a panel of pancreatic cancercell lines, the combination of erlotinib and Compound A was synergisticin the two mesenchymal cell lines and additive in the epithelial cellline tested (FIG. 11). For the set of ovarian cancer and HNSCC celllines tested, representative dose-response curves are shown (FIG. 12).Within the panel of ovarian and HNSCC cell lines, the combination oferlotinib and Compound A was synergistic in the majority of cell linestested (e.g. Igrov-1 Ovcar-3, HNSCC 1483) and additive in the remainder(e.g. CA-OV-3) although a strict correlation between mesenchymalphenotype and synergistic inhibition of proliferation was not observed.In no cell line tested was the combination of erlotinib and Compound Aantagonistic.

In a set of NSCLC and pancreatic cell lines, the combination of CompoundA and erlotinib was shown to potentiate the induction of apoptosis to agreater degree than either single agent in all but one cell line tested(FIG. 13). In that cell line, A1165, the level of apoptosis was equal tothat resulting from treatment with Compound A alone.

The combination enhanced apoptosis in both the mesenchymal andepithelial cell lines. Similarly, in a set of three ovarian cell lines,both erlotinib and Compound A increased apoptosis relative the DMSOtreated cells, and the combination of erlotinib and Compound A enhancedinduction of apoptosis to a greater degree than either single agent(FIG. 14).

Compound B has significant anti-proliferative activity in a panel ofHNSCC and ovarian cell lines (FIG. 14). The ability of 10 μM Compound Bto inhibit the growth of these cell lines is shown in FIG. 15. A maximalgrowth inhibition of greater than 50% was chosen as the criteria forhigh sensitivity and all but two of these can be categorized as verysensitive. When combined with erlotinib, Compound B synergisticallyinhibits cellular proliferation in the majority of cell lines tested.The effect of varying concentrations of Compound B on growth inhibitionin the presence and absence of erlotinib is shown for fourrepresentative cell lines in FIG. 16. Synergy was determined using theBliss model as described above. In no cell line tested was thecombination of erlotinib and Compound B antagonistic.

The combination of erlotinib and Compound B was also shown to enhanceapoptosis to a greater degree than either single agent in one ovarianand one HNSCC cell line (FIG. 17). In these cell lines erlotinib andCompound B were each capable of inducing a modest increase in apoptosisrelative to DMSO treated cells, and the combination of the two targetedagents resulted in a greater than 20 fold induction of apoptosis.

ABBREVIATIONS

EGF, epidermal growth factor; EGFR, epidermal growth factor receptor;EMT, epithelial-to-mesenchymal transition; MET,mesenchymal-to-epithelial transition; NSCL, non-small cell lung; NSCLC,non-small cell lung cancer; HNSCC, head and neck squamous cellcarcinoma; CRC, colorectal cancer; MBC, metastatic breast cancer; Brk,Breast tumor kinase (also known as protein tyrosine kinase 6 (PTK6));FCS, fetal calf serum; LC, liquid chromatography; MS, mass spectrometry;IGF-1, insulin-like growth factor-1; TGFat, transforming growth factoralpha; HB-EGF, heparin-binding epidermal growth factor; LPA,lysophosphatidic acid; IC₅₀, half maximal inhibitory concentration; pY,phosphotyrosine; wt, wild-type; PI3K, phosphatidyl inositol-3 kinase;GAPDH, glyceraldehyde 3-phosphate dehydrogenase; MAPK, mitogen-activatedprotein kinase; PDK-1,3-Phosphoinositide-Dependent Protein Kinase 1;Akt, also known as protein kinase B, is the cellular homologue of theviral oncogene v-Akt; mTOR, mammalian target of rapamycin; 4EBP 1,eukaryotic translation initiation factor-4E (mRNA cap-binding protein)Binding Protein-1, also known as PHAS-I; p70S6K, 70 kDa ribosomalprotein-S6 kinase; eIF4E, eukaryotic translation initiation factor-4E(mRNA cap-binding protein); Raf, protein kinase product of Raf oncogene;MEK, ERK kinase, also known as mitogen-activated protein kinase kinase;ERK, Extracellular signal-regulated protein kinase, also known asmitogen-activated protein kinase; PTEN, “Phosphatase and Tensinhomologue deleted on chromosome 10”, a phosphatidylinositol phosphatephosphatase; pPROTEIN, phospho-PROTEIN, “PROTEIN” can be any proteinthat can be phosphorylated, e.g. EGFR, ERK, S6 etc; PBS,Phosphate-buffered saline; TGI, tumor growth inhibition; WFI, Water forInjection; SDS, sodium dodecyl sulfate; ErbB2, “v-erb-b2 erythroblasticleukemia viral oncogene homolog 2”, also known as HER-2; ErbB3,“v-erb-b2 erythroblastic leukemia viral oncogene homolog 3”, also knownas HER-3; ErbB4, “v-erb-b2 erythroblastic leukemia viral oncogenehomolog 4”, also known as HER-4; FGFR, Fibroblast Growth FactorReceptor; DMSO, dimethyl sulfoxide.

INCORPORATION BY REFERENCE

All patents, published patent applications and other referencesdisclosed herein are hereby expressly incorporated herein by reference.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain, usingno more than routine experimentation, many equivalents to specificembodiments of the invention described specifically herein. Suchequivalents are intended to be encompassed in the scope of the followingclaims.

1-26. (canceled) 27: A method for treating tumors or tumor metastases in a patient, comprising administering to said patient simultaneously or sequentially a therapeutically effective amount of a combination of an EGFR kinase inhibitor and an agent that sensitizes tumor cells to the effects of EGFR kinase inhibitors, wherein said agent is an mTOR inhibitor that binds to and directly inhibits both mTORC1 and mTORC2 kinases, and is a compound represented by Formula (I),

or a pharmaceutically acceptable salt thereof, wherein: X₁, and X₂ are each independently N or C-(E¹)_(aa); X₅ is N, C-(E¹)_(aa), or N-(E¹)_(aa); X₃, X₄, X₆, and X₇ are each independently N or C; wherein at least one of X₃, X₆, X₄, X₅, X₆, and X₇ is independently N or N-(E¹)_(aa); R³ is C₀₋₁₀alkyl, cycloC₃₋₁₀alkyl, aminomethylcycloC₃₋₁₀alkyl, bicycloC₅₋₁₀alkyl, aryl, heteroaryl, aralkyl, heteroaralkyl, heterocyclyl or heterobicycloC₅₋₁₀alkyl any of which is optionally substituted by one or more independent G¹¹ substituents; Q¹ is -A(R¹)_(m)B(W)_(n) or —B(G¹¹)_(n)A(Y)_(m); A and B are respectively, 5 and 6 membered aromatic or heteroaromatic rings, fused together to form a 9-membered heteroaromatic system excluding 5-benzo[b]furyl and 3-indolyl; and excluding 2-indolyl, 2-benzoxazole, 2-benzothiazole, 2-benzimidazolyl, 4-aminopyrrolopyrimidin-5-yl, 4-aminopyrrolopyrimidin-6-yl, and 7-deaza-7-adenosinyl derivatives when X₁ and X₅ are CH, X₃, X₆ and X₇ are C, and X₂ and X₄ are N; or Q¹ is -A(R¹)_(m)A(Y)_(m), wherein each A is the same or different 5-membered aromatic or heteroaromatic ring, and the two are fused together to form an 8-membered heteroaromatic system; R¹ is independently, hydrogen, —N(C₀₋₈alkyl)(C₀₋₈alkyl), hydroxyl, halogen, oxo, aryl (optionally substituted with 1 or more R³¹ groups), hetaryl (optionally substituted with 1 or more R³¹ groups), C₁₋₆alkyl, —C₀₋₈alkylC₃₋₈cyclyoalkyl, —C₀₋₈alkyl-NR³¹¹S(O)₀₋₂R³²¹, —C₀₋₈alkyl-NR³¹¹S(O)₀₋₂NR³²¹R³³¹, —C₀₋₈alkyl-S(O)₀₋₂R³¹¹R³²¹, —C₀₋₈alkyl-NR³¹¹COR³²¹, —C₀₋₈alkyl-NR³¹¹CO₂R³²¹, —C₀₋₈alkyl-NR³¹¹CONR³²¹R³³¹, —C₀₋₈alkyl-CONR³¹¹R³²¹, —C₀₋₈alkyl-CON(R³¹¹)S(O)₀₋₂R³²¹, C₀₋₈alkyl-CO₂R³¹¹, —C₀₋₈alkyl-S(O)₀₋₂R³¹¹, C₀₋₈alkyl-O—C₁₋₈alkyl, —C₀₋₈alkyl-O—C₀₋₈alkylC₃₋₈cycloalkyl, —C₀₋₈alkyl-O—C₀₋₈alkylheterocycl, —C₀₋₈alkyl-O—C₀₋₈alkylaryl, —C₀₋₈alkylaryl, —C₀₋₈alkylhetaryl, —C₀₋₈alkylheterocyclyl, —C₀₋₈alkyl-O—C₀₋₈alkylhetaryl, —C₀₋₈alkyl-S—C₀₋₈alkyl, —C₀₋₈alkyl-S—C₀₋₈alkylC₃₋₈cyclyoalkyl, —C₀₋₈alkyl-S—C₀₋₈alkylheterocyclyl, —C₀₋₈alkyl-S—C₀₋₈alkylaryl, —C₀₋₈alkyl-S—C₀₋₈alkylhetaryl, —C₀₋₈alkyl-N(R³¹¹)—C₀₋₈alkyl, —C₀₋₈alkyl-N(R³¹¹)—C₀₋₈alkylC₃₋₈cycloalkyl, —C₀₋₈alkyl-N(R³¹¹)—C₀₋₈alkylheterocyclyl, —C₀₋₈alkyl-N(R³¹¹)—C₀₋₈alkylaryl, —C₀₋₈alkyl-N(R³¹¹)—C₀₋₈alkylhetaryl, —C₀₋₈alkyl-NR³¹¹R³²¹—C₂₋₈alkenyl, —C₂₋₈alkynyl, NO₂, CN, CF₃, OCF₃, OCHF₂; provided that Q¹ is not N-methyl-2-indolyl, N-(phenylsulfonyl)-2-indolyl, or N-tert-butoxycarbonyl W is independently, hydrogen, —N(C₀₋₈alkyl)(C₀₋₈alkyl), hydroxyl, halogen, oxo, aryl (optionally substituted with 1 or more R³¹ groups), hetaryl (optionally substituted with 1 or more R³¹ groups), C₁₋₆alkyl, —C₀₋₈alkylC₃₋₈cycloalkyl, —C₀₋₈alkyl-NR³¹²S(O)₀₋₂R³²², —C₀₋₈alkyl-NR³¹¹S(O)₀₋₂NR³²¹R³³¹—C₀₋₈alkyl-NR³¹¹CO₂R³²¹, —C₀₋₈alkyl-CON(R³¹¹)S(O)₀₋₂R³²¹C₀₋₈alkyl-S(O)₀₋₂NR³¹²R³²², —C₀₋₈alkyl-NR³¹²COR³²², —C₀₋₈alkyl-NR³¹²CONR³²²R³³², —C₀₋₈alkyl-CONR³¹²R³²², —C₀₋₈alkyl-CO₂R³¹², —C₀₋₈alkyl S(O)₀₋₂R³¹², —C₀₋₈alkyl-O—C₁₋₈alkyl, —C₀₋₈alkyl-O—C₀₋₈alkylcyclyl, —C₀₋₈alkyl-O—C₀₋₈alkylheterocycloalkyl, —C₀₋₈alkyl-O—C₀₋₈alkylaryl, —Oaryl, —C₀₋₈alkyl-O—C₀₋₈alkylhetaryl, —C₀₋₈alkylaryl, —C₀₋₈alkylhetaryl, —C₀₋₈alkylheterocyclyl, —C₀₋₈alkyl-S—C₀₋₈alkyl, —C₀₋₈alkyl-S—C₀₋₈alkylC₀₋₈cycloalkyl, —C₀₋₈alkyl-S—C₀₋₈alkylheterocycloalkyl, —C₀₋₈alkyl-S—C₀₋₈alkylaryl, —C₀₋₈alkyl-S—C₀₋₈alkylhetaryl, —C₀₋₈alkyl-N(R³¹²)—C₀₋₈alkyl, —C₀₋₈alkyl-N(R³¹²)—C₀₋₈alkylC₃₋₈cyclyoalkyl, —C₀₋₈alkyl-N(R³¹²)—C₀₋₈alkylheterocyclyoalkyl, —C₀₋₈alkyl-N(R³¹²)—C₀₋₈alkylaryl, —C₀₋₈alkyl-N(R³¹²)—C₀₋₈alkylhetaryl, —C₀₋₈alkyl-NR³¹²R³²², —C₂₋₈alkenyl, —C₂₋₈alkynyl, NO₂, CN, CF₃, OCF₃, OCHF₂; provided that Q¹ is not 4-benzyloxy-2-indolyl; Y is independently, hydrogen, —N(C₀₋₈alkyl)(C₀₋₈alkyl), hydroxyl, halogen, oxo, aryl (optionally substituted with 1 or more R³¹ groups), hetaryl (optionally substituted with 1 or more R³¹ groups), C₀₋₆alkyl, —C₀₋₈alkylC₃₋₈cycloalkyl, —C₀₋₈alkyl-NR³¹¹S(O)₀₋₂R³²¹, —C₀₋₈alkyl-NR³¹¹S(O)₀₋₂NR³²¹R³³¹, —C₀₋₈alkyl-NR³¹¹CO₂R³²¹, —C₀₋₈alkyl-CON(R³¹¹)S(O)₀₋₂R³²¹, —C₀₋₈alkyl-S(O)₀₋₂NR³¹¹R³²¹, —C₀₋₈alkyl-NR³¹¹COR³²¹, —C₀₋₈alkyl-NR³¹¹CONR³²¹R³³¹, —C₀₋₈alkyl-CONR³¹¹R³²¹, —C₀₋₈alkyl-CO₂R³¹¹, —C₀₋₈alkylS(O)₀₋₂R³¹¹, —C₀₋₈alkyl-O—C₁₋₈alkyl, —C₀₋₈alkyl-O—C₀₋₈alkylC₃₋₈cycloalkyl, —C₀₋₈alkyl-O—C₀₋₈alkylheterocycloalkyl, —C₀₋₈alkyl-O—C₀₋₈alkylaryl, —C₀₋₈alkyl-O—C₀₋₈alkylhetaryl, —C₀₋₈alkylaryl, —C₀₋₈alkylhetaryl, —C₀₋₈alkylheterocyclyl, C₀₋₈alkyl-S—C₀₋₈alkyl, —C₀₋₈alkyl-S—C₀₋₈alkylC₃₋₈cycloalkyl, —C₀₋₈alkyl-S—C₀₋₈alkylheterocycloalkyl, —C₀₋₈alkyl-S—C₀₋₈alkylaryl, —C₀₋₈alkyl-S—C₀₋₈alkylhetaryl, C₀₋₈alkyl-N(R³¹¹)—C₀₋₈alkyl, —C₀₋₈alkyl-N(R³¹¹)—C₀₋₈alkylC₃₋₈cycloalkyl, —C₀₋₈alkyl-N(R³¹¹)—C₀₋₈alkylheterocycloalkyl, —C₀₋₈alkyl-N(R³¹¹)—C₀₋₈alkylaryl, —C₀₋₈alkyl-N(R³¹¹)—C₀₋₈alkylhetaryl, —C₀₋₈alkyl-NR³¹¹R³²¹, —C₂₋₈alkenyl, —-C₂₋₈alkynyl, NO₂, CN, CF₃, OCF₃, OCHF₂; provided that Q¹ is not 2-carboxy-5-benzo[b]thiophenyl; G¹¹ is halo, oxo, —CF₃, —OCF₃, —OR³¹², —NR³¹²R³²², —C(O)R³¹², —C(O)C₃₋₈cycloalkyl, —CO₂C₃₋₈cycloalkyl, —CO₂R³¹², —C(═O)NR³¹²R³²², —NO₂, —CN, —S(O)₀₋₂R³¹², —SO₂NR³¹²R³²², NR³¹²(C═O)R³²², NR³¹²C(═O)OR³²², NR³¹²C(═O)NR³²²R³³², NR³¹²S(O)₀₋₂R³²², —C(═S)OR³¹², —C(═O)SR³¹², —NR³¹²C(═NR³²²)NR³³²R³⁴¹, —NR³¹²C(═NR³²²)OR³³², —NR³¹²C(═NR³²²)SR³³², —OC(═O)OR³¹², —OC(═O)NR³¹²R³²², —OC(═O)SR³¹², —SC(═O)OR³¹², —SC(═O)NR³¹²R³²², —P(O)OR³¹²OR³²², C₁₋₁₀alkylidene, C₀₋₁₀alkyl, C₂₋₁₀alkenyl, C₂₋₁₀alkynyl, C₁₋₁₀alkoxyC₁₋₁₀alkyl, —C₁₋₁₀alkoxyC₂₋₁₀alkenyl, —C₁₋₁₀alkoxyC₂₋₁₀alkynyl, —C₁₋₁₀alkylthioC₁₋₁₀alkyl, —C₁₋₁₀alkylthioC₂₋₁₀alkenyl, —C₁₋₁₀alkylthioC₂₋₁₀alkynyl, cycloC₃₋₈alkyl, cycloC₃₋₈alkenyl, -cycloC₃₋₈alkylC₁₋₁₀alkyl, -cycloC₃₋₈alkenylC₁₋₁₀alkyl, -cycloC₃₋₈alkylC₂₋₁₀alkenyl, -cycloC₃₋₈alkenylC₂₋₁₀alkenyl, -cycloC₃₋₈alkylC₂₋₁₀alkenyl, -cycloC₃₋₈alkenylC₂₋₁₀alkynyl, -heterocyclyl-C₀₋₁₀alkyl, heterocyclyl-C₂₋₁₀alkenyl, or -heterocyclyl-C₂₋₁₀alkynyl, any of which is optionally substituted with one or more independent halo, oxo, —CF₃, —OCF₃, —OR³¹³, —NR³¹³R³²³, —C(O)R³¹³, —CO₂R³¹³, —C(═O)NR³¹³R³²³, —NO₂, —CN, —S(O)₀₋₂R³¹³, —SO₂NR³¹³R³²³, —NR³¹³C(═O)R³²³, —NR³¹³C(═O)OR³²³, —NR³¹³C(═O)NR³²³R³³³—NR³¹³S(O)₀₋₂R³²³, —C(═S)OR³¹³—C(═O)SR³¹³—NR³¹³C(═NR³²³)NR³³³R³⁴², —NR³¹³C(═NR³²³)OR³³³, —NR³¹³C(═NR³²³)SR³³³, —OC(═O)OR³³³, —OC(═O)NR³¹³R³²³, —OC(═O)SR³¹³, —SC(═O)OR³¹³, —P(O)OR³¹³OR³²³, or —SC(═O)NR³¹³R³²³ substituents; or G¹¹ is aryl-C₀₋₁₀alkyl, aryl-C₂₋₁₀alkenyl, aryl-C₂₋₁₀alkynyl, hetaryl-C₀₋₁₀alkyl, hetaryl-C₂₋₁₀alkenyl, or hetaryl-C₂₋₁₀alkynyl, where the attachment point is from either the left or right as written, where any of which is optionally substituted with one or more independent halo, —CF₃, —OCF₃, —OR³¹³, —NR³¹³R³²³, —C(O)R³¹³, —CO₂R³¹³, —C(═O)NR³¹³R³²³, —NO₂, —CN, —S(O)₀₋₂R³¹³—SO₂NR³¹³R³²³, —NR³¹³C(═O)R³²³, —NR³¹³C(═O)OR³²³, —NR³¹³C(═O)NR³²³R³³³, —NR³¹³S(O)₀₋₂R³²³, —C(═S)OR³¹³, —C(═O)SR³¹³, —NR³²³C(═NR³¹³)NR³³³R³⁴², —NR³¹³C(═NR³²³)OR³³³, —NR³¹³C(═NR³²³)SR³³³, —OC(═O)OR³¹³, —OC(═O)NR³¹³R³²³, —OC(═O)SR³¹³, —SC(═O)OR³¹³, —P(O)OR³¹³OR³²³, or —SC(═O)NR³¹³R³²³ substituents; provided that G¹¹ is not N—CH₂CO₂H when R³ is 4-piperidinyl; R³¹, R³², R³³, R³¹¹, R³²¹, R³³¹, R³¹², R³²², R³³², R³⁴¹, R³¹³, R³²³, R³³³, and R³⁴², in each instance, is independently C₀₋₈alkyl optionally substituted with an aryl, heterocyclyl or hetaryl substituent, or C₀₋₈alkyl optionally substituted with 1-6 independent halo, —CON(C₀₋₈alkyl)(C₀₋₈alkyl), —CO(C₀₋₈alkyl), —OC₀₋₈alkyl, —Oaryl, —Ohetaryl, —Oheterocyclyl, —S(O)₀₋₂aryl, —S(O)₀₋₂hetaryl, —S(O)₀₋₂heterocyclyl, —S(O)₀₋₂C₀₋₈alkyl, —N(C₀₋₈alkyl)(C₀₋₈alkyl), —N(C₀₋₈alkyl)CON(C₀₋₈alkyl)(C₀₋₈alkyl)-N(C₀₋₈alkyl)CO(C₁₋₈alkyl), —N(C₀₋₈alkyl)CO(C₃₋₈cycloalkyl), —N(C₀₋₈alkyl)CO₂(C₁₋₈alkyl), —S(O)₁₋₂N(C₀₋₈alkyl)(C₀₋₈alkyl), —NR¹¹S(O)₁₋₂(C₀₋₈alkyl), —CON(C₃₋₈cycloalkyl)(C₃₋₈cycloalkyl), —CON(C₀₋₈alkyl)(C₃₋₈cycloalkyl), —N(C₃₋₈cycloalkyl)CON(C₀₋₈alkyl)(C₀₋₈alkyl), —N(C₃₋₈cycloalkyl)CON(C₃₋₈cycloalkyl)(C₀₋₈alkyl), —N(C₀₋₈alkyl)CON(C₃₋₈cycloalkyl)(C₀₋₈alkyl), —N(C₀₋₈alkyl)CO₂(C₃₋₈cycloalkyl), —N(C₃₋₈cycloalkyl)CO₂(C₃₋₈cycloalkyl), S(O)₁₋₂N(C₀₋₈alkyl)(C₃₋₈cycloalkyl), —NR¹¹S(O)₁₋₂(C₃₋₈cycloalkyl), C₂₋₈alkenyl, C₂₋₈alkynyl, CN, CF₃, OH, or optionally substituted aryl substituents; such that each of the above aryl, heterocyclyl, hetaryl, alkyl or cycloalkyl groups may be optionally, independently substituted with —N(C₀₋₈alkyl)(C₀₋₈alkyl), hydroxyl, halogen, oxo, aryl, hetaryl, C₀₋₆alkyl, C₀₋₈alkylcyclyl, —C₀₋₈alkyl-N(C₀₋₈alkyl)-S(O)₀₋₂—(C₀₋₈alkyl), —C₀₋₈alkyl-S(O)₀₋₂—N(C₀₋₈alkyl)(C₀₋₈alkyl), —C₀₋₈alkyl-N(C₀₋₈alkyl)CO(C₀₋₈alkyl), —C₀₋₈alkyl-N(C₀₋₈alkyl)CO—N(C₀₋₈alkyl)(C₀₋₈alkyl), —C₀₋₈alkyl-CO—N(C₀₋₈alkyl)(C₀₋₈alkyl), —C₁₋₈alkyl-CO₂—(C₀₋₈alkyl), —C₀₋₈alkylS(O)₀₋₂—(C₀₋₈alkyl), —C₀₋₈alkyl-O—C₁₋₈alkyl, —C₀₋₈alkyl-O—C₀₋₈alkylcyclyl, —C₀₋₈alkyl-O—C₀₋₈alkylheterocyclyl, —C₀₋₈alkyl-O—C₀₋₈alkylaryl, —C₀₋₈alkyl-O—C₀₋₈alkylhetaryl, —C₀₋₈alkyl-S—C₀₋₈alkyl, —C₀₋₈alkyl-S—C₀₋₈alkylcyclyl, —C₀₋₈alkyl-S—C₀₋₈alkylheterocyclyl, —C₀₋₈alkyl-S—C₀₋₈alkylaryl, —C₀₋₈alkyl-S—C₀₋₈alkylhetaryl, —C₀₋₈alkyl-N(C₀₋₈alkyl)-C₀₋₈alkyl, —C₀₋₈alkyl-N(C₀₋₈alkyl)-C₀₋₈alkylcyclyl, —C₀₋₈alkyl-N(C₀₋₈alkyl)-C₀₋₈alkylheterocyclyl, —C₀₋₈alkyl-N(C₀₋₈alkyl)-C₀₋₈alkylaryl, —C₀₋₈alkyl-N(C₀₋₈alkyl)-C₀₋₈alkylhetaryl, C₂₋₈alkenyl, C₂₋₈alkynyl, NO₂, CN, CF₃, OCF₃, OCHF₂, —C₀₋₈alkyl-C₃₋₈cycloalkyl, —C₀₋₈alkyl-O—C₀₋₈alkyl, —C₀₋₈alkyl-N(C₀₋₈alkyl)(C₀₋₈alkyl), —C₀₋₈alkyl-S(O)₀₋₂—C₀₋₈alkyl, or heterocyclyl optionally substituted with 1-4 independent C₀₋₈alkyl, cyclyl, or substituted cyclyl substituents; E¹ in each instance is independently halo, —CF₃, —OCF₃, —OR², —NR³¹R³², —C(═O)R³¹, —CO₂R³¹, —CONR³¹R³², —NO₂, —CN, —S(O)₀₋₂R³¹—S(O)₀₋₂NR³¹R³², —NR³¹C(═O)R³², —NR³¹C(═O)OR³², —NR³¹C(═O)NR³²R³³, —NR³¹S(O)₀₋₂R³², —C(═S)OR³¹, —C(═O)SR³¹, —NR³¹C(═NR³²)NR³³R³¹, —NR³¹C(═NR³²)OR³³, —NR³¹C(═NR³¹)SR³¹, —OC(═O)OR³¹, —OC(═O)NR³¹R³², —OC(═O)SR³¹, —SC(═O)OR³¹, —SC(═O)NR³¹R³², C₀₋₁₀alkyl, C₂₋₁₀alkenyl, C₂₋₁₀alkynyl, —C₁₋₁₀alkoxyC₁₋₁₀alkyl, —C₁₋₁₀alkoxyC₂₋₁₀alkenyl, —C₁₋₁₀alkoxyC₂₋₁₀alkynyl, —C₁₋₁₀alkylthioC₁₋₁₀alkyl, —C₁₋₁₀alkylthioC₂₋₁₀alkenyl, —C₁₋₁₀alkylthioC₂₋₁₀alkynyl, cycloC₃₋₈alkyl, cycloC₃₋₈alkenyl, -cycloC₃₋₈alkylC₁₋₁₀alkyl, -cycloC₃₋₈alkenylC₁₋₁₀alkyl, -cycloC₃₋₈alkylC₂₋₁₀alkenyl, -cycloC₃₋₈alkenylC₂₋₁₀alkenyl, -cycloC₃₋₈alkylC₂₋₁₀alkynyl-cycloC₃₋₈alkenylC₂₋₁₀alkynyl, -heterocyclyl-C₀₋₁₀alkyl, -heterocyclyl-C₂₋₁₀alkenyl, or -heterocyclyl-C₂₋₁₀alkynyl, any of which is optionally substituted with one or more independent halo, oxo, —CF₃, —OCF₃, —OR³¹, —NR³¹R³², —C(═O)R³¹, —CO₂R³¹, —C(═O)NR³¹R³², —NO₂, —CN, —S(═O)₀₋₂, R³¹, —SO₂NR³¹, —NR³¹C(═O)R³², —NR³¹C(═O)OR³¹, —NR³¹C(═O)NR³²R³³, —NR³¹S(O)₀₋₂R³¹, —C(═S)OR³¹, —C(═O)SR³¹, —NR³¹C(═NR³²)NR³³R³¹, —NR³¹C(═NR³²)OR³³, —NR³¹C(═NR³²)SR³³, —OC(═O)OR³¹, —OC(═O)NR³¹R³², —OC(═O)SR³¹, —SC(═O)OR³¹, or —SC(═O)NR³¹R³² substituents; or E¹ in each instance is independently aryl-C₀₋₁₀alkyl, aryl-C₂₋₁₀alkenyl, aryl-C₂₋₁₀alkynyl, hetaryl-C₀₋₁₀alkyl, hetaryl-C₂₋₁₀alkenyl, or hetaryl-C₂₋₁₀alkynyl, where the attachment point is from either the left or right as written, where any of which is optionally substituted with one or more independent halo, —CF₃, —OCF₃, —OR³¹, —NR¹³R³², —C(O)R³¹, —CO₂R³¹, —C(═O)NR³¹R³², —NO₂, —CN, —S(O)₀₋₂R³¹, —S(O)₀₋₂NR³¹R³², —NR³¹C(═O)R³², —NR³¹C(═O)OR³², —NR C(═O)NR³²R³³, —NR³¹S(O)₀₋₂R³², —C(═S)OR³¹, —C(═O)SR³¹, —NR³¹C(═NR³²)NR³³R³¹, —NR³¹C(═NR³²)OR³³, —NR¹³C(═NR³²)SR³³, —OC(═O)OR³¹, —OC(═O)NR³¹R³², —OC(═O)SR³¹, —SC(═O)OR³¹, or —SC(═O)NR³¹R³² substituents; in the cases of —NR³¹R³², —NR³¹¹R³²¹, —NR³¹²R³²², —NR³³²R³⁴¹, —NR³¹³R³²³, and —NR³²³R³³³, the respective R³¹ and R³², R³¹¹ and R³²¹, R³¹² and R³²², R³³¹ and R³⁴¹, R³¹³ and R³²³, and R³²³ and R³³³ are optionally taken together with the nitrogen atom to which they are attached to form a 3-10 membered saturated or unsaturated ring; wherein said ring in each instance independently is optionally substituted by one or more independent —N(C₀₋₈alkyl)(C₀₋₈alkyl), hydroxyl, halogen, oxo, aryl, hetaryl, C₀₋₆alkyl, —C₀₋₈alkylC₃₋₈cycloalkyl, —C₀₋₈alkyl-N(C₀₋₈alkyl)S(O)₀₋₂C₀₋₈alkyl, —C₀₋₈alkyl-N(C₀₋₈alkyl)S(O)₀₋₂N(C₀₋₈alkyl)(C₀₋₈alkyl), —C₀₋₈alkyl-N(C₀₋₈alkyl)CO₂(C₀₋₈alkyl), —C₀₋₈alkyl-CON((C₀₋₈alkyl))S(O)₀₋₂(C₀₋₈alkyl), —C₀₋₈alkyl-S(O)₀₋₂N(C₀₋₈alkyl)(C₀₋₈alkyl), —C₀₋₈alkyl-N(C₀₋₈alkyl)CO(C₀₋₈alkyl), —C₀₋₈alkyl-N(C₀₋₈alkyl)CON(C₀₋₈alkyl)(C₀₋₈alkyl), —C₀₋₈alkyl-CON(C₀₋₈alkyl)(C₀₋₈alkyl), —C₀₋₈alkyl-CO₂(C₀₋₈alkyl), —C₀₋₈alkylS(O)₀₋₂(C₀₋₈alkyl), —C₀₋₈alkyl-O—C₀₋₈alkyl, —C₀₋₈alkyl-O—C₀₋₈alkylcyclyl, —C₀₋₈alkyl-O—C₀₋₈alkylheterocycloalkyl, —C₀₋₈alkyl-O—C₀₋₈alkylaryl, —Oaryl, —C₀₋₈alkyl-O—C₀₋₈alkylhetaryl, —C₀₋₈alkyl-S—C₀₋₈alkyl, —C₀₋₈alkyl-S—C₀₋₈alkylC₃₋₈cycloalkyl-C₀₋₈alkyl-S—C₀₋₈alkylheterocycloalkyl, —C₀₋₈alkyl-S—C₀₋₈alkylaryl, —C₀₋₈alkyl-S—C₀₋₈alkylhetaryl, —C₀₋₈alkyl-N(C₀₋₈alkyl)-C₀₋₈alkyl, —C₀₋₈alkyl-N(C₀₋₈alkyl)-C₀₋₈alkylC₃₋₈cycloalkyl, —C₀₋₈alkyl-N(C₀₋₈alkyl)-C₀₋₈alkylheterocycloalkyl, —C₀₋₈alkyl-N(C₀₋₈alkyl)-C-alkylaryl, —C₀₋₈alkyl-N(C₀₋₈alkyl)-C₀₋₈alkylhetaryl, —C₀₋₈alkyl-N(C₀₋₈alkyl)(C₀₋₈alkyl), C₂₋₈alkenyl, C₂₋₈alkynyl, NO₂, CN, CF₃, OCF₃, or OCHF₂ substituents; wherein said ring in each instance independently optionally includes one or more heteroatoms other than the nitrogen; m is 0, 1, 2, or 3; n is 0, 1, 2, 3, or 4; aa is 0 or 1; and provided that Formula I is not trans-4-[8-amino-1-(7-chloro-4-hydroxy-1H-indol-2-yl)imidazo[1,5-a]pyrazin-3-yl]cyclohexanecarboxylic acid, cis-3-[8-amino-1-(7-chloro-1H-indol-2-yl)imidazo[1,5-a]pyrazin-3-yl]cyclobutanecarboxylic acid, trans-4-{8-amino-1-[7-(3-isopropyl)phenyl-1H-indol-2-yl]imidazo[1,5-a]pyrazin-3-yl}cyclohexanecarboxylic acid or trans-4-{8-amino-1-[7-(2,5-dichloro)phenyl-1H-indol-2-yl]imidazo[1,5-a]pyrazin-3-yl}cyclohexanecarboxylic acid. 28: The method of claim 27, wherein the patient is a human that is being treated for cancer. 29: The method of claim 27, wherein the EGFR kinase inhibitor and mTOR inhibitor are co-administered to the patient in the same formulation. 30: The method of claim 27, wherein the EGFR kinase inhibitor and mTOR inhibitor are co-administered to the patient in different formulations. 31: The method of claim 27, wherein the EGFR kinase inhibitor and mTOR inhibitor are co-administered to the patient by the same route. 32: The method of claim 27, wherein the EGFR kinase inhibitor and mTOR inhibitor are co-administered to the patient by different routes. 33: The method of claim 27, wherein the EGFR kinase inhibitor is a small organic molecule, an antibody or an antibody fragment that binds specifically to the EGFR. 34: The method of claim 27, wherein the EGFR kinase inhibitor comprises erlotinib, or a salt thereof. 35: The method of claim 27, additionally comprising administering to said patient one or more other anti-cancer agents. 36: The method of claim 27, wherein the administering to the patient is simultaneous. 37: The method of claim 27, wherein the administering to the patient is sequential. 38: The method of claim 27, wherein the cells of the tumors or tumor metastases have high sensitivity to growth inhibition by EGFR kinase inhibitors as single agents. 39: The method of claim 27, wherein the cells of the tumors or tumor metastases have low sensitivity to growth inhibition by EGFR kinase inhibitors as single agents. 40: The method of claim 27, wherein the cells of the tumors or tumor metastases have not undergone any form of EMT. 41: The method of claim 27, wherein the cells of the tumors or tumor metastases have undergone an EMT. 42: A method for the treatment of cancer, comprising administering to a subject in need of such treatment an amount of the EGFR kinase inhibitor, or a pharmaceutically acceptable salt thereof; and an amount of an mTOR inhibitor that binds to and directly inhibits both mTORC1 and mTORC2 kinases, or a pharmaceutically acceptable salt thereof; wherein at least one of the amounts is administered as a sub-therapeutic amount, and is a compound represented by Formula (I),

or a pharmaceutically acceptable salt thereof, wherein: X₁, and X₂ are each independently N or C-(E¹)_(aa); X₅ is N, C-(E¹)_(aa), or N-(E¹)_(aa); X₃, X₄, X₆, and X₇ are each independently N or C; wherein at least one of X₃, X₄, X₅, X₆, and X₇ is independently N or N-(E¹)_(aa); R³ is C₀₋₁₀alkyl, cycloC₃₋₁₀alkyl, aminomethylcycloC₃₋₁₀alkyl, bicycloC₅₋₁₀alkyl, aryl, heteroaryl, aralkyl, heteroaralkyl, heterocyclyl or heterobicycloC₅₋₁₀alkyl any of which is optionally substituted by one or more independent G¹¹ substituents; Q¹ is -A(R¹)_(m)B(W)_(n) or —B(G¹¹)_(n)A(Y)_(m); A and B are respectively, 5 and 6 membered aromatic or heteroaromatic rings, fused together to form a 9-membered heteroaromatic system excluding 5-benzo[b]furyl and 3-indolyl; and excluding 2-indolyl, 2-benzoxazole, 2-benzothiazole, 2-benzimidazolyl, 4-aminopyrrolopyrimidin-5-yl, 4-aminopyrrolopyrimidin-6-yl, and 7-deaza-7-adenosinyl derivatives when X₁ and X₅ are CH, X₃, X₆ and X₇ are C, and X₂ and X₄ are N; or Q¹ is -A(R¹)_(m)A(Y)_(m), wherein each A is the same or different 5-membered aromatic or heteroaromatic ring, and the two are fused together to form an 8-membered heteroaromatic system; R¹ is independently, hydrogen, —N(C₀₋₈alkyl)(C₀₋₈alkyl), hydroxyl, halogen, oxo, aryl (optionally substituted with 1 or more R³¹ groups), hetaryl (optionally substituted with 1 or more R³¹ groups), C₁₋₆alkyl, —C₀₋₈alkylC₃₋₈cycloalkyl, —C₀₋₈alkyl-NR³¹¹S(O)₀₋₂R³²¹, —C₀₋₈alkyl-NR³¹¹S(O)₀₋₂NR³²¹R³³¹, —C₀₋₈alkyl-S(O)₀₋₂NR³¹¹R³²¹, —C₀₋₈alkyl-NR³¹¹COR³²¹, —C₀₋₈alkyl-NR³¹¹CO₂R³²¹, —C₀₋₈alkyl-NR³¹¹CONR³²¹R³³¹, —C₀₋₈alkyl-CONR³¹¹R³²¹, —C₀₋₈alkyl-CON(R³¹¹)S(O)₀₋₂R³²¹, —C₀₋₈alkyl-CO₂R³¹¹, —C₀₋₈alkyl-S(O)₀₋₂R³¹¹, —C₀₋₈alkyl-O—C₁₋₈alkyl, —C₀₋₈alkyl-O—C₀₋₈alkylC₃₋₈cycloalkyl, —C₀₋₈alkyl-O—C₀₋₈alkylheterocyclyl, —C₀₋₈alkyl-O—C₀₋₈alkylaryl, —C₀₋₈alkylaryl, —C₀₋₈alkylhetaryl, —C₀₋₈alkylheterocyclyl, —C₀₋₈alkyl-O—C₀₋₈alkylhetaryl, —C₀₋₈alkyl-S—C₀₋₈alkyl, —C₀₋₈alkyl-S—C₀₋₈alkylC₃₋₈cycloalkyl, —C₀₋₈alkyl-S—C₀₋₈alkylheterocyclyl, —C₀₋₈alkyl-S—C₀₋₈alkylaryl, —C₀₋₈alkyl-S—C₀₋₈alkylhetaryl, —C₀₋₈alkyl-N(R³¹¹)—C₀₋₈alkyl, —C₀₋₈alkyl-N(R³¹¹)—C₀₋₈alkylC₃₋₈cycloalkyl, —C₀₋₈alkyl-N(R³¹¹)—C₀₋₈alkylheterocyclyl, —C₀₋₈alkyl-N(R³¹¹)—C₀₋₈alkylaryl, —C₀₋₈alkyl-N(R³¹¹)—C₀₋₈alkylhetaryl, —C₀₋₈alkyl-NR³¹¹R³²¹, —C₂₋₈alkenyl, —C₂₋₈alkynyl, NO₂, CN, CF₃, OCF₃, OCHF₂ provided that Q¹ is not N-methyl-2-indolyl, N-(phenylsulfonyl)-2-indolyl, or N-tert-butoxycarbonyl W is independently, hydrogen, —N(C₀₋₈alkyl)(C₀₋₈alkyl), hydroxyl, halogen, oxo, aryl (optionally substituted with 1 or more R³¹ groups), hetaryl (optionally substituted with 1 or more R³¹ groups), C₁₋₆alkyl, —C₀₋₈alkylC₃₋₈cycloalkyl, —C₀₋₈alkyl-NR³¹²S(O)₀₋₂R³²², —C₀₋₈alkyl-NR³¹¹S(O)₀₋₂NR³²¹R³³¹, —C₀₋₈alkyl-NR³¹¹CO₂R³²¹, —C₀₋₈alkyl-CON(R³¹¹)S(O)₀₋₂R³²¹, —C₀₋₈alkyl-S(O)₀₋₂NR³¹²R³²², —C₀₋₈alkyl-NR³¹²COR³²², —C₀₋₈alkyl-NR³¹²CONR³²²R³³², —C₀₋₈alkyl-CONR³¹²R³²², —C₀₋₈alkyl-CO₂R³¹², —C₀₋₈alkylS(O)₀₋₂R³¹², —C₀₋₈alkyl-O—C₁₋₈alkyl, —C₀₋₈alkyl-O—C₀₋₈alkylcyclyl, —C₀₋₈alkyl-O—C₀₋₈alkylheterocycloalkyl, —C₀₋₈alkyl-O—C₀₋₈alkylaryl, —Oaryl, —C₀₋₈alkyl-O—C₀₋₈alkylhetaryl, —C₀₋₈alkylaryl, —C₀₋₈alkylhetaryl, —C₀₋₈alkylheterocyclyl, —C₀₋₈alkyl-S—C₀₋₈alkyl, —C₀₋₈alkyl-S—C₀₋₈alkylC₃₋₈cycloalkyl, —C₀₋₈alkyl-S—C₀₋₈alkylheterocycloalkyl, —C₀₋₈alkyl-S—C₀₋₈alkylaryl, —C₀₋₈alkyl-S—C₀₋₈alkylhetaryl, —C₀₋₈alkyl-N(R³¹²)—C₀₋₈alkyl, —C₀₋₈alkyl-N(R³¹²)—C₀₋₈alkylC₀₋₈cycloalkyl, —C₀₋₈alkyl-N(R³¹²)—C₀₋₈alkylheterocycloalkyl, —C₀₋₈alkyl-N(R³¹²)—C₀₋₈alkylaryl, —C₀₋₈alkyl-N(R³¹²)—C₀₋₈alkylhetaryl, —C₀₋₈alkyl-NR³¹²R³²², —C₂₋₈alkenyl, —C₂₋₈alkynyl, NO₂, CN, CF₃, OCF₃, OCHF₂; provided that Q¹ is not 4-benzyloxy-2-indolyl; Y is independently, hydrogen, —N(C₀₋₈alkyl)(C₀₋₈alkyl), hydroxyl, halogen, oxo, aryl (optionally substituted with 1 or more R³¹ groups), hetaryl (optionally substituted with 1 or more R³¹ groups), C₀₋₆alkyl, —C₀₋₈alkylC₃₋₈cycloalkyl, —C₀₋₈alkyl-NR³¹¹S(O)₀₋₂R³²¹, —C₀₋₈alkyl-NR³¹¹S(O)₀₋₂NR³²¹R³³¹, —C₀₋₈alkyl-NR³¹¹CO₂R³²¹, —C₀₋₈alkyl-CON(R³¹¹)S(O)₀₋₂R³²¹, —C₀₋₈alkyl-S(O)₀₋₂NR³¹¹R³²¹, —C₀₋₈alkyl-NR³¹¹COR³²¹, —C₀₋₈alkyl-NR³¹¹CONR³²¹R³³¹, —C₀₋₈alkyl-CONR³¹¹R³²¹, —C₀₋₈alkyl-CO₂R³¹¹, —C₀₋₈alkylS(O)₀₋₂R³¹¹, —C₀₋₈alkyl-O—C₁₋₈alkyl, —C₀₋₈alkyl-O—C₀₋₈alkylC₃₋₈cycloalkyl, —C₀₋₈alkyl-O—C₀₋₈alkylheterocycloalkyl, —C₀₋₈alkyl-O—C₀₋₈alkylaryl, —C₀₋₈alkyl-O—C₀₋₈alkylhetaryl, —C₀₋₈alkylaryl, —C₀₋₈alkylhetaryl, —C₀₋₈alkylheterocyclyl, —C₀₋₈alkyl-S—C₀₋₈alkyl, —C₀₋₈alkyl-S—C₀₋₈alkylC₃₋₈cycloalkyl, —C₀₋₈alkyl-S—C₀₋₈alkylheterocycloalkyl, —C₀₋₈alkyl-S—C₀₋₈alkylaryl, —C₀₋₈alkyl-S—C₀₋₈alkylhetaryl, —C₀₋₈alkyl-N(R³¹¹)—C₀₋₈alkyl, —C₀₋₈alkyl-N(R³¹¹)—C₀₋₈alkylC₃₋₈cycloalkyl, —C₀₋₈alkyl-N(R³¹¹)—C₀₋₈alkylheterocycloalkyl, —C₀₋₈alkyl-N(R³¹¹)—C₀₋₈alkylaryl, —C₀₋₈alkyl-N(R³¹¹)—C₀₋₈alkylhetaryl, —C₀₋₈alkyl-NR³¹¹R³²¹—C₂₋₈alkenyl, —C₂₋₈alkynyl, NO₂, CN, CF₃, OCF₃, OCHF₂; provided that Q¹ is not 2-carboxy-5-benzo[b]thiophenyl; G¹¹ is halo, oxo, —CF₃, —OCF₃, —OR³¹², —NR³¹²R³²², —C(O)R³¹², —C(O)C₃₋₈cycloalkyl, —CO₂C₃₋₈cycloalkyl, —CO₂R³¹², —C(═O)NR³¹²R³²², —NO₂, —CN, —S(O)₀₋₂R³¹²—SO₂NR³¹²R³²², NR³¹²(C═O)R³²², NR³¹²C(═O)OR³²², NR³¹²C(═O)NR³²²R³³², NR³¹²S(O)₀₋₂R³²²—C(═S)OR³¹², —C(═O)SR³¹², —NR³¹²C(═NR³²²)NR³³²R³⁴¹, —NR³¹²C(═NR³²²)OR³³², —NR³¹²C(═NR³²²)SR³³², —OC(═O)OR³¹², —OC(═O)NR³¹²R³²², —OC(═O)SR³¹², —SC(═O)OR³¹², —SC(═O)NR³¹²R³²², —P(O)OR³¹²OR³²², C₁₋₁₀alkylidene, C₀₋₁₀alkyl, C₂₋₁₀alkenyl, C₂₋₁₀alkynyl, —C₁₋₁₀alkoxyC₁₋₁₀alkyl, —C₁₋₁₀alkoxyC₂₋₁₀alkenyl, —C₁₋₁₀alkoxyC₂₋₁₀alkynyl, —C₁₋₁₀alkylthioC₁₋₁₀alkyl, —C₁₋₁₀alkylthioC₂₋₁₀alkenyl, —C₁₋₁₀alkylthioC₂₋₁₀alkynyl, cycloC₃₋₈alkyl, cycloC₃₋₈alkenyl, -cycloC₃₋₈alkylC₁₋₁₀alkyl, -cycloC₃₋₈alkenylC₁₋₁₀alkyl, -cycloC₃₋₈alkylC₂₋₁₀alkenyl, -cycloC₃₋₈alkenylC₂₋₁₀alkenyl, -cycloC₃₋₈alkylC₂₋₁₀alkynyl, -cycloC₃₋₈alkenylC₂₋₁₀alkynyl, -heterocyclyl-C₁₋₁₀alkyl, heterocyclyl-C₂₋₁₀alkenyl, or -heterocyclyl-C₂₋₁₀alkynyl, any of which is optionally substituted with one or more independent halo, oxo, —CF₃, —OCF₃, —OR³¹³, —NR³¹³R³²³, —C(O)R³¹³, —CO₂R³¹³, —C(═O)NR³¹³R³²³—NO₂, —CN, —S(O)₀₋₂R³¹³, —SO₂NR³¹³R³²³, —NR³¹³C(═O)R³²³, —NR³¹³C(═O)OR³²³, —NR³¹³C(═O)NR³²³R³³³, —NR³¹³S(O)₀₋₂R³²³, —C(═S)OR³¹³, —C(═O)SR³¹³, —NR³¹³C(═NR³²³)NR³³³R³⁴², —NR³¹³C(═NR³²³)OR³³³, —NR³¹³C(═NR³²³SR)SR³³³, —OC(═O)OR³³³, —OC(═O)NR³¹³R³²³, —OC(═O)SR³¹³, —SC(═O)OR³¹³, —P(O)OR³¹³OR³²³, or —SC(═O)NR³¹³R³²³ substituents; or G¹¹ is aryl-C₀₋₁₀alkyl, aryl-C₂₋₁₀alkenyl, aryl-C₂₋₁₀alkynyl, hetaryl-C₀₋₁₀alkyl, hetaryl-C₂₋₁₀alkenyl, or hetaryl-C₂₋₁₀alkynyl, where the attachment point is from either the left or right as written, where any of which is optionally substituted with one or more independent halo, —CF₃, —OCF₃, —OR³¹³, —NR³¹³R³²³, —C(O)R³¹³, —CO₂R³¹³, —C(═O)NR³¹³R³²³, —NO₂, —CN, —S(O)₀₋₂R³¹³, —SO₂NR³¹³R³²³, —NR³¹³C(═O)R³²³, —NR³¹³C(═O)OR³²³, —NR³¹³C(═O)NR³²³R³³³, —NR³¹³S(O)₀₋₂R³²³, —C(═S)OR³¹³, —C(═O)SR³¹³, —NR³²³C(═NR³¹³)NR³³³R³⁴², —NR³¹³C(═NR³²³)OR³³³, —NR³¹³C(═NR³²³)SR³³³, —OC(═O)OR³¹³, —OC(═O)NR³¹³R³²³, —OC(═O)SR³¹³, —SC(═O)OR³¹³, —P(O)OR³¹³OR³²³, or —SC(═O)NR³¹³R³²³ substituents; provided that G¹¹ is not N—CH₂CO₂H when R³ is 4-piperidinyl; R³¹, R³², R³³, R³¹¹, R³²¹, R³³¹, R³¹², R³²², R³³², R³⁴¹, R³¹³, R³²³, R³³³, and R³⁴², in each instance, is independently C₀₋₈alkyl optionally substituted with an aryl, heterocyclyl or hetaryl substituent, or C₀₋₈alkyl optionally substituted with 1-6 independent halo, —CON(C₀₋₈alkyl)(C₀₋₈alkyl), —CO(C₀₋₈alkyl), —OC₀₋₈alkyl, —Oaryl, —Ohetaryl, —Oheterocyclyl, —S(O)₀₋₂aryl, —S(O)₀₋₂hetaryl, —S(O)₀₋₂heterocyclyl, —S(O)₀₋₂C₀₋₈alkyl, —N(C₀₋₈alkyl)(C₀₋₈alkyl), —N(C₀₋₈alkyl)CON(C₀₋₈alkyl)(C₀₋₈alkyl), —N(C₀₋₈alkyl)CO(C₁₋₈alkyl), —N(C₀₋₈alkyl)CO(C₃₋₈cycloalkyl), —N(C₀₋₈alkyl)CO₂(C₁₋₈alkyl)-S(O)₁₋₂N(C₀₋₈alkyl)(C₀₋₈alkyl), —NR¹¹S(O)₁₋₂(C₀₋₈alkyl), —CON(C₃₋₈cycloalkyl)(C₃₋₈cycloalkyl), —CON(C₀₋₈alkyl)(C₃₋₈cycloalkyl), —N(C₃₋₈cycloalkyl)CON(C₀₋₈alkyl)(C₀₋₈alkyl), —N(C₃₋₈cycloalkyl)CON(C₃₋₈cycloalkyl)(C₀₋₈alkyl), —N(C₀₋₈alkyl)CON(C₃₋₈cyclyoalkyl)(C₀₋₈alkyl), —N(C₀₋₈alkyl)CO₂(C₃₋₈cycloalkyl), —N(C₃₋₈cyclyoalkyl)CO₂(C₃₋₈cycloalkyl), S(O)₁₋₂N(C₀₋₈alkyl)(C₃₋₈cycloalkyl), —NR¹¹S(O)₁₋₂(C₃₋₈cyclyoalkyl), C₂₋₈alkenyl, C₂₋₈alkynyl, CN, CF₃, OH, or optionally substituted aryl substituents; such that each of the above aryl, heterocyclyl, hetaryl, alkyl or cyclyoalkyl groups may be optionally, independently substituted with —N(C₀₋₈alkyl)(C₀₋₈alkyl), hydroxyl, halogen, oxo, aryl, hetaryl, C₀₋₆alkyl, C₀₋₈alkylcyclyl, —C₀₋₈alkyl-N(C₀₋₈alkyl)S(O)₀₋₂—(C₀₋₈alkyl), —C₀₋₈alkyl-S(O)₀₋₂—N(C₀₋₈alkyl)(C₀₋₈alkyl), —C₀₋₈alkyl-N(C₀₋₈alkyl)CO(C₀₋₈alkyl), —C₀₋₈alkyl-N(C₀₋₈alkyl)CO—N(C₀₋₈alkyl)(C₀₋₈alkyl), —C₀₋₈alkyl-CO—N(C₀₋₈alkyl)(C₀₋₈alkyl), —C₁₋₈alkyl-CO₂—(C₀₋₈alkyl), —C₀₋₈alkylS(O)₀₋₂—(C₀₋₈alkyl), —C₀₋₈alkyl-O—C₁₋₈alkyl, —C₀₋₈alkyl-O—C₀₋₈alkylcyclyl, —C₀₋₈alkyl-O—C₀₋₈alkylheterocyclyl, —C₀₋₈alkyl-O—C₀₋₈alkylaryl, —C₀₋₈alkyl-O—C₀₋₈alkylhetaryl, —C₀₋₈alkyl-S—C₀₋₈alkyl, —C₀₋₈alkyl-S—C₀₋₈alkylcyclyl, —C₀₋₈alkyl-S—C₀₋₈alkylheterocyclyl, —C₀₋₈alkyl-S—C₀₋₈alkylaryl, —C₀₋₈alkyl-S—C₀₋₈alkylhetaryl, —C₀₋₈alkyl-N(C₀₋₈alkyl)-C₀₋₈, —C₀₋₈alkyl-N(C₀₋₈alkyl)-C₀₋₈alkylcyclyl, —C₀₋₈alkyl-N(C₀₋₈alkyl)-C₀₋₈alkylheterocyclyl, —C₀₋₈alkyl-N(C₀₋₈alkyl)-C₀₋₈alkylaryl, —C₀₋₈alkyl-N(C₀₋₈alkyl)-C₀₋₈alkylhetaryl, C₂₋₈alkenyl, C₂₋₈alkynyl, NO₂, CN, CF₃, OCF₃, OCHF₂, —C₀₋₈alkyl-C₃₋₈cycloalkyl, —C₀₋₈alkyl-O—C₀₋₈alkyl, —C₀₋₈alkyl-N(C₀₋₈alkyl)(C₀₋₈alkyl), —C₀₋₈alkyl-SO₀₋₂C₀₋₈alkyl, or heterocyclyl optionally substituted with 1-4 independent C₀₋₈alkyl, cyclyl, or substituted cyclyl substituents; E¹ in each instance is independently halo, —CF₃, —OCF₃, —OR², —NR³¹R³², —C(═O)R³¹, —CO₂R³¹, —CONR³¹R³², —NO₂, —CN, —S(O)₀₋₂R³¹, —S(O)₀₋₂NR³¹R³², —NR³¹C(═O)R³², —NR³¹C(═O)OR³², —NR³¹C(═O)NR³²R³³, —NR³¹S(O)₀₋₂R³², —C(═S)OR³¹, —C(═O)SR³¹, —NR³¹C(═NR³²)NR³³R³¹, —NR³¹C(═NR³²)OR³³, —NR³¹C(═NR³¹)SR³¹, —OC(═O)OR³¹, —OC(═O)NR³¹R³², —OC(═O)SR³¹, —SC(═O)OR³¹, —SC(═O)NR³¹R³², C₀₋₁₀alkyl, C₂₋₁₀alkenyl, C₂₋₁₀alkynyl, —C₁₋₁₀alkoxyC₁₋₁₀alkyl, —C₁₋₁₀alkoxyC₂₋₁₀alkenyl, —C₁₋₁₀alkoxyC₂₋₁₀alkynyl, —C₁₋₁₀alkylthioC₁₋₁₀alkyl, —C₁₋₁₀alkylthioC₂₋₁₀alkenyl, —C₁₋₁₀alkylthioC₂₋₁₀alkynyl, cycloC₃₋₈alkyl, cycloC₃₋₈alkenyl, -cycloC₃₋₈alkylC₁₋₁₀alkyl, -cycloC₃₋₈alkenylC₁₋₁₀alkyl, -cycloC₃₋₈alkylC₂₋₁₀alkenyl, -cycloC₃₋₈alkenylC₂₋₁₀alkenyl, -cycloC₃₋₈alkylC₂₋₁₀alkynyl, -cycloC₃₋₈alkenylC₂₋₁₀alkynyl, -heterocyclyl-C₀₋₁₀alkyl, -heterocyclyl-C₂₋₁₀alkenyl, or -heterocyclyl-C₂₋₁₀alkynyl, any of which is optionally substituted with one or more independent halo, oxo, —CF₃, —OCF₃, —OR³¹, —NR³¹R³², —C(═O)R³¹, —CO₂R³¹, —C(═O)NR³¹R³², —NO₂, —CN, —S(═O)₀₋₂R³¹, —SO₂NR³¹, —NR³¹C(═O)R³², —NR³¹C(═O)OR³¹, —NR³¹C(═O)NR³²R³³, —NR³¹S(O)₀₋₂R³¹, —C(═S)OR³¹, —C(═O)SR³¹, —NR³¹C(═NR³²)NR³³R³¹, —NR³¹C(═NR³²)OR³³, —NR³¹C(═NR³²)SR³³, —OC(═O)OR³¹, —OC(═O)NR³¹R³², —OC(═O)SR³¹, —SC(═O)OR³¹, or —SC(═O)NR³¹R³² substituents; or E¹ in each instance is independently aryl-C₀₋₁₀alkyl, aryl-C₂₋₁₀alkenyl, aryl-C₂₋₁₀alkynyl, hetaryl-C₀₋₁₀alkyl, hetaryl-C₂₋₁₀alkenyl, or hetaryl-C₂₋₁₀alkynyl, where the attachment point is from either the left or right as written, where any of which is optionally substituted with one or more independent halo, —CF₃, —OCF₃, —OR³¹, —NR³¹R³², —C(O)R³¹, —CO₂R³¹, —C(═O)NR³¹R³², —NO₂, —CN, —S(O)₀₋₂R³¹, —S(O)₀₋₂NR³¹R³², —NR³¹C(═O)R³², —NR³¹C(═O)OR³², —NR³¹C(═O)NR³²R³³, —NR³¹S(O)₀₋₂R³², —C(═S)OR³¹, —C(═O)SR³¹, —NR³¹C(═NR³²)NR³³R³¹, —NR³¹C(═NR³²)OR³³, —NR³¹C(═NR³²)SR³³, —OC(═O)OR³¹, —OC(═O)NR³¹R³², —OC(═O)SR³¹, —SC(═O)OR³¹, or —SC(═O)NR³¹R³² substituents; in the cases of —NR¹³R³², —NR³¹¹R³²¹, —NR³¹²R³²², —NR³³²R³⁴¹, —NR³¹³R³²³, and —NR³²³R³³³, the respective R³¹ and R³², R³¹¹ and R³²¹, R³¹² and R³²², R³³¹ and R³⁴¹, R³¹³ and R³²³, and R³²³ and R³³³ are optionally taken together with the nitrogen atom to which they are attached to form a 3-10 membered saturated or unsaturated ring; wherein said ring in each instance independently is optionally substituted by one or more independent —N(C₀₋₈alkyl)(C₀₋₈alkyl), hydroxyl, halogen, oxo, aryl, hetaryl, C₀₋₆alkyl, —C₀₋₈alkylC₃₋₈cycloalkyl, —C₀₋₈alkyl-N(C₀₋₈alkyl)S(O)₀₋₂C₀₋₈alkyl, —C₀₋₈alkyl-N(C₀₋₈alkyl)S(O)₀₋₂N(C₀₋₈alkyl)(C₀₋₈alkyl), —C₀₋₈alkyl-N(C₀₋₈alkyl)CO₂(C₀₋₈alkyl), —C₀₋₈alkyl-CON((C₀₋₈alkyl))S(O)₀₋₂(C₀₋₈alkyl), —C₀₋₈alkyl-S(O)₀₋₂N(C₀₋₈alkyl)(C₀₋₈alkyl), —C₀₋₈alkyl-N(C₀₋₈alkyl)CO(C₀₋₈alkyl), —C₀₋₈alkyl-N(C₀₋₈alkyl)CON(C₀₋₈alkyl)(C₀₋₈alkyl), —C₀₋₈alkyl-CON(C₀₋₈alkyl)(C₀₋₈alkyl), —C₀₋₈alkyl CO₂(C₀₋₈alkyl), —C₀₋₈alkyl S(O)₀₋₂(C₀₋₈alkyl), —C₀₋₈alkyl-O—C₀₋₈alkyl-C₀₋₈alkyl-O—C₀₋₈alkylcyclyl, —C₀₋₈alkyl-O—C₀₋₈alkylheterocyclyoalkyl, —C₀₋₈alkyl-O—C₀₋₈alkylaryl, —Oaryl, —C₀₋₈alkyl-O—C₀₋₈alkylhetaryl, —C₀₋₈alkyl-S—C₀₋₈alkyl, —C₀₋₈alkyl-S—C₀₋₈alkylC₃₋₈cycloalkyl, —C₀₋₈alkyl-S—C₀₋₈alkylheterocycloalkyl, —C₀₋₈alkyl-S—C₀₋₈alkylaryl, —C₀₋₈alkyl-S—C₀₋₈alkylhetaryl, C₀₋₈alkyl-N(C₀₋₈alkyl)-C₀₋₈alkyl, —C₀₋₈alkyl-N(C₀₋₈alkyl)-C₀₋₈alkylC₃₋₈cycloalkyl, —C₀₋₈alkyl-N(C₀₋₈alkyl)-C₀₋₈alkylheterocycloalkyl, —C₀₋₈alkyl-N(C₀₋₈alkyl), —C₀₋₈alkylaryl, —C₀₋₈alkyl-N(C₀₋₈alkyl)-C₀₋₈alkylhetaryl, —C₀₋₈alkyl-N(C₀₋₈alkyl)(C₀₋₈alkyl) C₂₋₈alkenyl, C₂₋₈alkynyl, NO₂, CN, CF₃, OCF₃, or OCHF₂, substituents; wherein said ring in each instance independently optionally includes one or more heteroatoms other than the nitrogen; m is 0, 1, 2, or 3: n is 0, 1, 2, 3, or 4: aa is 0 or 1; and provided that Formula I is not trans-4-[8-amino-1-(7-chloro-4-hydroxy-1H-indol-2-yl)imidazo[1,5-a]pyrazin-3-yl]cyclohexanecarboxylic acid, cis-3-[8-amino-1-(7-chloro-1H-indol-2-yl)imidazo[1,5-a]pyrazin-3-yl]cyclobutanecarboxylic acid, trans-4-{8-amino-1-[7-(3-isopropyl)phenyl-1H-indol-2-yl]imidazo[1,5-a]pyrazin-3-yl}cyclohexanecarboxylic acid or trans-4-{8-amino-1-[7-(2,5-dichloro)phenyl-1H-indol-2-yl]imidazo[1,5-a]pyrazin-3-yl}cyclohexanecarboxylic acid. 43: The method of claim 42, wherein the EGFR kinase inhibitor comprises erlotinib, or a salt thereof. 44: The method of claim 42, additionally comprising administering to said subject one or more other anti-cancer agents. 45: A method for treating tumors or tumor metastases in a patient, comprising administering to said patient simultaneously or sequentially a synergistically effective therapeutic amount of a combination of an EGFR kinase inhibitor and an mTOR inhibitor that binds to and directly inhibits both mTORC1 and mTORC2 kinases, and is a compound represented by Formula (I),

or a pharmaceutically acceptable salt thereof, wherein: X₁, and X₂ are each independently N or C-(E¹)_(aa); X₅ is N, C-(E¹)_(aa), or N-(E¹)_(aa); X₃, X₄, X₆, and X₇ are each independently N or C; wherein at least one of X₃, X₄, X₅, X₆, and X₇ is independently N or N-(E¹)_(aa); R³ is C₀₋₁₀alkyl, cycloC₃₋₁₀alkyl, aminomethylcycloC₃₋₁₀alkyl, bicycloC₅₋₁₀alkyl, aryl, heteroaryl, aralkyl, heteroaralkyl, heterocyclyl or heterobicycloC₅₋₁₀alkyl any of which is optionally substituted by one or more independent G¹¹ substituents; Q¹ is -A(R¹)_(m)B(W)_(n) or —B(G¹¹)_(n)A(Y)_(m); A and B are respectively, 5 and 6 membered aromatic or heteroaromatic rings, fused together to form a 9-membered heteroaromatic system excluding 5-benzo[b]furyl and 3-indolyl; and excluding 2-indolyl, 2-benzoxazole, 2-benzothiazole, 2-benzimidazolyl, 4-aminopyrrolopyrimidin-5-yl, 4-aminopyrrolopyrimidin-6-yl, and 7-deaza-7-adenosinyl derivatives when X₁ and X₅ are CH, X₃, X₆ and X₇ are C, and X₂ and X₄ are N; or Q¹ is -A(R¹)_(m)A(Y)_(m), wherein each A is the same or different 5-membered aromatic or heteroaromatic ring, and the two are fused together to form an 8-membered heteroaromatic system; R¹ is independently, hydrogen, —N(C₀₋₈alkyl)(C₀₋₈alkyl), hydroxyl, halogen, oxo, aryl (optionally substituted with 1 or more R³¹ groups), hetaryl (optionally substituted with 1 or more R³¹ groups), C₁₋₆alkyl, —C₀₋₈alkylC₃₋₈cycloalkyl, C₀₋₈alkyl-NR³¹¹S(O)₀₋₂R³²¹, —C₀₋₈alkyl-NR³¹¹S(O)₀₋₂NR³²¹R³³¹, —C₀₋₈alkyl-S(O)₀₋₈NR³¹¹R³²¹, —C₀₋₈alkyl-NR³¹¹COR³²¹, —C₀₋₈alkyl-NR³¹¹CO₂R³²¹, —C₀₋₈alkyl-NR³¹¹CONR³²¹R³³¹, —C₀₋₈alkyl-CONR³¹¹R³²¹, —C₀₋₈alkyl-CON(R³¹¹)S(O)₀₋₂R³²¹, —C₀₋₈alkyl-CO₂R³¹¹, —C₀₋₈alkyl-S(O)₀₋₂R³¹¹, —C₀₋₈alkyl-O—C₁₋₈alkyl, —C₀₋₈alkyl-O—C₀₋₈alkylC₃₋₈cycloalkyl, —C₀₋₈alkyl-O—C₀₋₈alkylheterocyclyl, —C₀₋₈alkyl-O—C₀₋₈alkylaryl, —C₀₋₈alkylaryl, —C₀₋₈alkylhetaryl, —C₀₋₈alkylheterocyclyl, —C₀₋₈alkyl-O—C₀₋₈alkylhetaryl, —C₀₋₈alkyl-S—C₀₋₈alkyl, —C₀₋₈alkyl-S—C₀₋₈alkylC₃₋₈cycloalkyl, —C₀₋₈alkyl-S—C₀₋₈alkylheterocyclyl, —C₀₋₈alkyl-S—C₀₋₈alkylaryl, —C₀₋₈alkyl-S—C₀₋₈alkylhetaryl, —C₀₋₈alkyl-N(R³¹¹)—C₀₋₈alkyl, —C₀₋₈alkyl-N(R³¹¹)—C₀₋₈alkylC₃₋₈cyclyoalkyl, —C₀₋₈alkyl-N(R³¹¹)—C₀₋₈alkylheterocyclyl, —C₀₋₈alkyl-N(R³¹¹)—C₀₋₈alkylaryl, —C₀₋₈alkyl-N(R³¹¹)—C₀₋₈alkylhetaryl, —C₀₋₈alkyl-NR³¹¹R³²¹, —C₂₋₈alkenyl, —C₂₋₈alkynyl, NO₂, CN, CF₃, OCF₃, OCHF₂; provided that Q¹ is not N-methyl-2-indolyl, N-(phenylsulfonyl)-2-indolyl, or N-tert-butoxycarbonyl W is independently, hydrogen, —N(C₀₋₈alkyl)(C₀₋₈alkyl), hydroxyl, halogen, oxo, aryl (optionally substituted with 1 or more R³¹ groups), hetaryl (optionally substituted with 1 or more R³¹ groups), C₁₋₆alkyl, —C₀₋₈alkylC₃₋₈cycloalkyl, C₀₋₈alkyl-NR³¹²S(O)₀₋₂R³²², —C₀₋₈alkyl-NR³¹¹S(O)₀₋₂NR³²¹R³³¹, —C₀₋₈alkyl-NR³¹¹CO₂R³²¹, —C₀₋₈alkyl-CON(R³¹¹)S(O)₀₋₂R³²¹, —C₀₋₈alkyl-S(O)₀₋₂NR³¹²R³²², —C₀₋₈alkyl-NR³¹²COR³²², —C₀₋₈alkyl-NR³¹¹CONR³²²R³³², —C₀₋₈alkyl-CONR³¹²R³²², —C₀₋₈alkyl-CO₂R³¹², —C₀₋₈alkylS(O)₀₋₂R³¹², —C₀₋₈alkyl-O—C₁₋₈alkyl, —C₀₋₈alkyl-O—C₀₋₈alkylcyclyl, —C₀₋₈alkyl-O—C₀₋₈alkylheterocyclyoalkyl, —C₀₋₈alkyl-O—C₀₋₈alkylaryl, —Oaryl, —C₀₋₈alkyl-O—C₀₋₈alkylhetaryl, —C₀₋₈alkylaryl, —C₀₋₈alkylhetaryl, —C₀₋₈alkylheterocyclyl, —C₀₋₈alkyl-S—C₀₋₈alkyl, —C₀₋₈alkyl-S—C₀₋₈alkylC₃₋₈cycloalkyl, —C₀₋₈alkyl-S—C₀₋₈alkylheterocycloalkyl, —C₀₋₈alkyl-S—C₀₋₈alkylaryl, —C₀₋₈alkyl-S—C₀₋₈alkylhetaryl, —C₀₋₈alkyl-N(R³¹²)—C₀₋₈alkyl, —C₀₋₈alkyl-N(R³¹²)—C₀₋₈alkylC₃₋₈cyclyoalkyl, —C₀₋₈alkyl-N(R³¹²)—C₀₋₈alkylheterocycloalkyl, —C₀₋₈alkyl-N(R³¹²)—C₀₋₈alkylaryl, —C₀₋₈alkyl-N(R³¹²)—C₀₋₈alkylhetaryl, —C₀₋₈alkyl-NR³¹²R³²², —C₂₋₈alkenyl, —C₂₋₈alkynyl, NO₂, CN, CF₃, OCF₃, OCHF₂; provided that Q¹ is not 4-benzyloxy-2-indolyl; Y is independently, hydrogen, —N(C₀₋₈alkyl)(C₀₋₈alkyl), hydroxyl, halogen, oxo, aryl (optionally substituted with 1 or more R³¹ groups), hetaryl (optionally substituted with 1 or more R³¹ groups), C₀₋₆alkyl, —C₀₋₈alkylC₃₋₈cycloalkyl, —C₀₋₈alkyl-NR³¹¹SO₀₋₂R³²¹, —C₀₋₈alkyl-NR³¹¹S(O)₀₋₂NR³²¹R³³¹, —C₀₋₈alkyl-NR³¹¹CO₂R³²¹, —C₀₋₈alkyl-CON(R³¹¹)S(O)₀₋₂R³²¹, —C₀₋₈alkyl-S(O)₀₋₂NR³¹¹R³²¹, —C₀₋₈alkyl-NR³¹¹COR³²¹, —C₀₋₈alkyl-NR³¹¹CONR³²¹R³³¹, —C₀₋₈alkyl-CONR³¹¹R³²¹, —C₀₋₈alkyl-CO₂R³¹¹, —C₀₋₈alkylS(O)₀₋₂R³¹¹, —C₀₋₈alkyl-O—C₁₋₈alkyl, —C₀₋₈alkyl-O—C₀₋₈alkylC₃₋₈cycloalkyl, —C₀₋₈alkyl-O—C₀₋₈alkylheterocycloalkyl, —C₀₋₈alkyl-O—C₀₋₈alkylaryl, —C₀₋₈alkyl-O—C₀₋₈alkylhetaryl, —C₀₋₈alkylaryl, —C₀₋₈alkylhetaryl, —C₀₋₈alkylheterocyclyl, —C₀₋₈alkyl-S—C₀₋₈alkyl, —C₀₋₈alkyl-S—C₀₋₈alkylC₃₋₈cycloalkyl, —C₀₋₈alkyl-S—C₀₋₈alkylheterocycloalkyl, —C₀₋₈alkyl-S—C₀₋₈alkaryl, —C₀₋₈alkyl-S—C₀₋₈alkylhetaryl, —C₀₋₈alkyl-N(R³¹¹)—C₀₋₈alkyl, —C₀₋₈alkyl-N(R³¹¹)—C₀₋₈alkylC₃₋₈cycloalkyl, —C₀₋₈alkyl-N(R³¹¹)—C₀₋₈alkylheterocycloalkyl, —C₀₋₈alkyl-N(R³¹¹)—C₀₋₈alkylaryl, —C₀₋₈alkyl-N(R³¹¹)—C₀₋₈alkylhetaryl, —C₀₋₈alkyl-NR³¹¹R³²¹, —C₀₋₈alkenyl, —C₂₋₈alkynyl, NO₂, CN, CF₃, OCF₃, OCHF₂; provided that Q¹ is not 2-carboxy-5-benzo[b]thiophenyl; G¹¹ is halo, oxo, —CF₃, —OCF₃, —OR³¹², —NR³¹²R³²², —C(O)R³¹²—CO)₃₋₈cycloalkyl, —CO₂C₃₋₈cycloalkyl, —CO₂R³¹², —C(═O)NR³¹²R³²², —NO₂, —CN, —S(O)₀₋₂R³¹², —SO₂NR³¹²R³²², NR³¹²(C═O)R³²², NR³¹²C(═O)OR³²², NR³¹²C(═O)NR³²²R³³², NR³¹²S(O)₀₋₂R³²², C(═S)OR³¹², —C(═O)SR³¹², —NR³¹²C(═NR³²²)NR³³²R³⁴¹, —NR³¹²C(═NR³²²)OR³³², —NR³¹²C(═NR³²²)SR³³², —OC(═O)OR³¹², —OC(═O)NR³¹²R³²², —OC(═O)SR³¹², —SC(═O)OR³¹², —SC(═O)NR³¹²R³²², —P(O)OR³¹²OR³²², C₁₋₁₀alkylidene, C₀₋₁₀alkyl, C₂₋₁₀alkenyl, C₂₋₁₀alkynyl, —C₁₋₁₀alkoxyC₁₋₁₀alkyl, —C₁₋₁₀alkoxyC₂₋₁₀alkenyl, —C₁₋₁₀alkoxyC₂₋₁₀alkynyl, —C₁₋₁₀alkylthioC₁₋₁₀alkyl, —C₁₋₁₀alkylthioC₂₋₁₀alkenyl, —C₁₋₁₀alkylthioC₂₋₁₀alkynyl, cycloC₃₋₈alkyl, cycloC₃₋₈alkenyl, -cycloC₃₋₈alkylC₁₋₁₀alkyl, -cycloC₃₋₈alkenylC₁₋₁₀alkyl, -cycloC₃₋₈alkylC₂₋₁₀alkenyl, -cycloC₃₋₈alkenylC₂₋₁₀alkenyl, -cycloC₃₋₈alkylC₂₋₁₀alkynyl, -cycloC₃₋₈alkenylC₂₋₁₀alkynyl, -heterocyclyl-C₀₋₁₀alkyl, heterocyclyl-C₂₋₁₀alkenyl, or -heterocyclyl-C₂₋₁₀alkynyl, any of which is optionally substituted with one or more independent halo, oxo, —CF₃, —OCF₃, —OR³¹³, —NR³¹³R³²³, —C(O)R³¹³, —CO₂R³¹³, —C(═O)NR³¹³R³²³, —NO₂, —CN, —S(O)₀₋₂R³¹³, —SO₂NR³¹³R³²³, —NR³¹³C═)R³²³, NR³¹³C(═O)OR³²³, —R³¹³C(═O)R³²³R³³³, —NR³¹³S(O)₀₋₂R³²³, —C(═S)OR³¹³, —C(═O)SR³¹³, —NR³¹³C(═NR³²³)NR³³³R³⁴², —NR³¹³C(═NR³²³)OR³³³, —NR³¹³C(═NR³²³)SR³³³, —OC(═O)OR³³³, —OC(═O)NR³¹³R³²³, —OC(═O)SR³¹³, —SC(═O)OR³¹³, —P(O)OR³¹³R³²³, or —SC(═O)NR³¹³R³²³ substituents; or G¹¹ is aryl-C₀₋₁₀alkyl, aryl-C₂₋₁₀alkenyl, aryl-C₂₋₁₀alkynyl, hetaryl-C₀₋₁₀alkyl, hetaryl-C₂₋₁₀alkenyl, or hetaryl-C₂₋₁₀alkynyl, where the attachment point is from either the left or right as written, where any of which is optionally substituted with one or more independent halo, —CF₃, —OCF₃, —OR³¹³, —NR³¹³R³²³, —C(O)R³¹³, —CO₂R³¹³, —C(═O)NR³¹³R³²³, —NO₂, —CN, —S(O)₀₋₂R³¹³, —SO₂NR³¹³R³²³, —NR³¹¹C(═O)R³²³, —NR³¹³C(═O)OR³²³, —NR³¹³C(═O)NR³²³R³³³, —NR³¹³S(O)₀₋₂R³³³, —C(═S)OR³¹³, —C(═O)SR³¹³, —NR³²³C(═NR³¹³)NR³³³R³⁴², —NR³¹³C(═NR³²³)OR³³³, —NR³¹³C(═NR³²³)SR³³³, —OC(═O)OR³¹³, —OC(═O)NR³¹³R³²³, —OC(═O)SR³¹³, —SC(═O)OR³¹³, —P(O)OR³¹³OR³²³, or —SC(═O)NR³¹³R³²³ substituents; provided that G¹¹ is not N—CH₂CO₂H when R³ is 4-piperidinyl; R³¹, R³², R³³, R³¹¹, R³²¹, R³³¹, R³¹², R³²², R³³², R³⁴¹, R³¹³, R³²³, R³³³, and R³⁴², in each instance, is independently C₀₋₈alkyl optionally substituted with an aryl, heterocyclyl or hetaryl substituent, or C₀₋₈alkyl optionally substituted with 1-6 independent halo, —CON(C₀₋₈alkyl)(C₀₋₈alkyl), —CO(C₀₋₈alkyl), —OC₀₋₈alkyl, —Oaryl, —Ohetaryl, —Oheterocyclyl, —S(O)₀₋₂aryl, —S(O)₀₋₂hetaryl, —SO₀₋₂heterocyclyl, S(O)₀₋₂C₀₋₈alkyl, —N(C₀₋₈alkyl)(C₀₋₈alkyl), —N(C₀₋₈alkyl)CON(C₀₋₈alkyl)(C₀₋₈alkyl), —N(C₀₋₈alkyl)CO(C₁₋₈alkyl), —N(C₀₋₈alkyl)CO(C₃₋₈cycloalkyl), —N(C₀₋₈alkyl)CO₂(C₁₋₈alkyl), —S(O)₁₋₂N(C₀₋₈alkyl)(C₀₋₈alkyl), —NR¹¹S(O)₁₋₂(C₀₋₈alkyl), —CON(C₃₋₈cycloalkyl)(C₃₋₈cycloalkyl), —CON(C₀₋₈alkyl)(C₃₋₈cyclyoalkyl), —N(C₃₋₈cycloalkyl)CON(C₀₋₈alkyl)(C₀₋₈alkyl), —N(C₃₋₈cycloalkyl)CON(C₃₋₈cycloalkyl)(C₀₋₈alkyl), —N(C₀₋₈alkyl)CON(C₃₋₈cycloalkyl)(C₀₋₈alkyl), —N(C₀₋₈alkyl)CO₂(C₃₋₈cycloalkyl), —N(C₃₋₈cycloalkyl)CO₂(C₃₋₈cycloalkyl), S(O)₁₋₂N(C₀₋₈alkyl)(C₃₋₈cycloalkyl), —NR¹¹S(O)₁₋₂(C₃₋₈cycloalkyl), C₂₋₈alkenyl, C₂₋₈alkynyl, CN, CF₃, OH, or optionally substituted aryl substituents; such that each of the above aryl, heterocyclyl, hetaryl, alkyl or cycloalkyl groups may be optionally, independently substituted with —N(C₀₋₈alkyl)(C₀₋₈alkyl), hydroxyl, halogen, oxo, aryl, hetaryl, C₀₋₆alkyl, C₀₋₈alkylcyclyl, —C₀₋₈alkyl-N(C₀₋₈alkyl)-S(O)₀₋₂C₀₋₈alkyl), —C₀₋₈alkyl-S(O)₀₋₂—N(C₀₋₈alkyl)(C₀₋₈alkyl), —C₀₋₈alkyl-N(C₀₋₈alkyl)CO(C₀₋₈alkyl), —C₀₋₈alkyl-N(C₀₋₈alkyl)CO—N(C₀₋₈alkyl)(C₀₋₈alkyl), —C₀₋₈alkyl-CO—N(C₀₋₈alkyl)(C₀₋₈alkyl), —C₁₋₈alkyl-CO₂—(C₀₋₈alkyl), —C₀₋₈alkylS(O)₀₋₂—(C₀₋₈alkyl), —C₀₋₈alkyl-O—C₁₋₈alkyl, —C₀₋₈alkyl-O—C₀₋₈alkylcyclyl, —C₀₋₈alkyl-O—C₀₋₈alkylheterocyclyl, —C₀₋₈alkyl-O—C₀₋₈alkylaryl, —C₀₋₈alkyl-O—C₀₋₈alkylhetaryl, —C₀₋₈alkyl-S—C₀₋₈alkyl, —C₀₋₈alkyl-S—C₀₋₈alkylcyclyl, —C₀₋₈alkyl-S—C₀₋₈alkylheterocyclyl, —C₀₋₈alkyl-S—C₀₋₈alkylaryl, —C₀₋₈alkyl-S—C₀₋₈alkylhetaryl, —C₀₋₈alkyl-N(C₀₋₈alkyl), —C₀₋₈alkyl, —C₀₋₈alkyl-N(C₀₋₈alkyl)-C₀₋₈alkylcyclyl, —C₀₋₈alkyl-N(C₀₋₈alkyl)-C₀₋₈alkylheterocyclyl, —C₀₋₈alkyl-N(C₀₋₈alkyl)-C₀₋₈alkylaryl, —C₀₋₈alkyl-N(C₀₋₈alkyl)-C₀₋₈alkylhetaryl, C₂₋₈alkenyl, C₂₋₈alkynyl, NO₂, CN, CF₃, OCF₃, OCHF₂, —C₀₋₈alkyl-C₃₋₈cycloalkyl, —C₀₋₈alkyl-O—C₀₋₈alkyl, —C₀₋₈alkyl-N(C₀₋₈alkyl)(C₀₋₈alkyl), —C₀₋₈alkyl-S(O)₀₋₂—C₀₋₈alkyl, or heterocyclyl optionally substituted with 1-4 independent C₀₋₈alkyl, cyclyl, or substituted cyclyl substituents; E¹ in each instance is independently halo, —CF₃, —OCF₃, —OR², —NR³¹R³², —C(═O)R³¹, —CO₂R³¹, —CONR³¹R³², —NO₂, —CN, —S(O)₀₋₂NR³¹R³², —NR³¹C(═O)R³², —NR³¹C(═O)OR³², —NR³¹C(═O)NR³²R³³, —NR³¹S(O)₀₋₂R³², —C(═S)OR³¹, —C(═O)SR³¹, —NR³¹C(═NR³²)NR³³R³¹, —NR³¹C(═NR³²)OR³³, —NR³¹C(═NR³¹)SR³¹, —OC(═O)OR³¹, —OC(═O)NR³¹R³², —OC(═O)SR³¹, —SC(═O)OR³¹, —SC(═O)NR³¹R³², C₀₋₁₀alkyl, C₂₋₁₀alkenyl, C₂₋₁₀alkynyl, —C₁₋₁₀alkoxyC₁₋₁₀alkyl, —C₁₋₁₀alkoxyC₂₋₁₀alkenyl, —C₁₋₁₀alkoxyC₂₋₁₀alkynyl, —C₁₋₁₀alkylthioC₁₋₁₀alkyl, —C₁₋₁₀alkylthioC₂₋₁₀alkenyl, —C₁₋₁₀alkylthioC₂₋₁₀alkynyl, cycloC₃₋₈alkyl, cycloC₃₋₈alkenyl, -cycloC₃₋₈alkylC₁₋₁₀alkyl, -cycloC₃₋₈alkenylC₁₋₁₀alkyl, -cycloC₃₋₈alkylC₂₋₁₀alkenyl, -cycloC₃₋₈alkenylC₂₋₁₀alkenyl, -cycloC₃₋₈alkylC₂₋₁₀alkynyl, -cycloC₃₋₈alkenylC₂₋₁₀alkynyl, -heterocyclyl-C₀₋₁₀alkyl, -heterocyclyl-C₂₋₁₀alkenyl, or -heterocyclyl-C₂₋₁₀alkynyl, any of which is optionally substituted with one or more independent halo, oxo, —CF₃, —OCF₃, —OR³¹, —NR³¹R³², —C(═O)R³¹, —CO₂R³¹, —C(═O)NR³¹R³², —NO₂, —CN, —S(═O)₀₋₂R³¹—SO₂NR³¹, —NR³¹C(═O)R³², —NR³¹C(═O)OR³¹, —NR³¹C(═O)NR³²R³³, —NR³¹S(O)₀₋₂R³¹, —C(═S)OR³¹, —C(═O)SR³¹, —NR³¹C(═NR³²)NR³³R³¹, —NR³¹C(═NR³²)OR³³, —NR³¹C(═NR³²)SR³³, —OC(═O)OR³¹, —OC(═O)NR³¹R³², —OC(═O)SR³¹, —SC(═O)OR³¹, or —SC(═O)NR³¹R³² substituents; or E¹ in each instance is independently aryl-C₀₋₁₀alkyl, aryl-C₂₋₁₀alkenyl, aryl-C₂₋₁₀alkynyl, hetaryl-C₀₋₁₀alkyl, hetaryl-C₂₋₁₀alkenyl, or hetaryl-C₂₋₁₀alkynyl, where the attachment point is from either the left or right as written, where any of which is optionally substituted with one or more independent halo, —CF, —OCF₃, —OR³¹, —NR³¹R³², —C(O)R³¹, —CO₂R³¹, —C(═O)NR³¹R³², —NO₂, —CN, —S(O)₀₋₂R³¹, —S(O)₀₋₂NR³¹R³², —NR³¹C(═O)R³², —NR³¹C(═O)OR³², —NR³¹C(═O)NR³²R³³, —NR³¹S(O)₀₋₂R³², —C(═S)OR³¹, —C(═O)SR³¹, —NR³¹C(═NR³²)NR³³R³¹, —NR³¹C(═NR³²)OR³³, —NR³¹C(═NR³²)SR³³, —OC(═O)OR³¹, —OC(═O)NR³¹R³², —OC(═O)SR³¹, —SC(═O)OR³¹, or —SC(═O)NR³¹R³² substituents; in the cases of —NR³¹R³², —NR³¹¹R³²¹, —NR³¹²R²²², —NR³³²R³⁴¹, —NR³¹³R³²³, and —NR³²³R³³³, the respective R³¹ and R³², R³¹¹ and R³²¹, R³¹² and R³²², R³³¹ and R³⁴¹, R³¹³ and R³²³, and R³²³ and R³³³ are optionally taken together with the nitrogen atom to which they are attached to form a 3-10 membered saturated or unsaturated ring; wherein said ring in each instance independently is optionally substituted by one or more independent —N(C₀₋₈alkyl)(C₀₋₈alkyl), hydroxyl, halogen, oxo, aryl, hetaryl, C₀₋₆alkyl, —C₀₋₈alkylC₃₋₈cycloalkyl, —C₀₋₈alkyl-N(C₀₋₈alkyl)S(O)₀₋₂C₀₋₈alkyl, —C₀₋₈alkyl-N(C₀₋₈alkyl)S(O)₀₋₂N(C₀₋₈alkyl)(C₀₋₈alkyl), —C₀₋₈alkyl-N(C₀₋₈alkyl)CO₂(C₀₋₈alkyl), —C₀₋₈—CON((C₀₋₈alkyl))S(O)₀₋₂(C₀₋₈alkyl), —C₀₋₈alkyl-S(O)₀₋₂N(C₀₋₈alkyl)(C₀₋₈alkyl), —C₀₋₈alkyl-N(C₀₋₈alkyl)CO(C₀₋₈alkyl), —C₀₋₈alkyl-N(C₀₋₈alkyl)CON(C₀₋₈alkyl)(C₀₋₈alkyl), —C₀₋₈alkyl-CON(C₀₋₈alkyl)(C₀₋₈alkyl), —C₀₋₈alkyl-CO₂(C₀₋₈alkyl), —C₀₋₈alkylS(O)₀₋₂(C₀₋₈alkyl), —C₀₋₈alkyl-O—C₀₋₈alkyl, —C₀₋₈alkyl-O—C₀₋₈alkylcyclyl, —C₀₋₈alkyl-O—C₀₋₈alkylheterocycloalkyl, —C₀₋₈alkyl-O—C₀₋₈alkylaryl, —Oaryl, —C₀₋₈alkyl-O—C₀₋₈alkylhetaryl, —C₀₋₈alkyl-S—C₀₋₈alkyl, —C₀₋₈alkyl-S—C₀₋₈alkylC₃₋₈cycloalkyl, —C₀₋₈alkyl-S—C₀₋₈alkylheterocycloalkyl, —C₀₋₈alkyl-S—C₀₋₈alkylaryl, —C₀₋₈alkyl-S—C₀₋₈alkylhetaryl, —C₀₋₈alkyl-N(C₀₋₈alkyl)-C₀₋₈alkyl, —C₀₋₈alkyl-N(C₀₋₈alkyl)-C₀₋₈alkylC₃₋₈cycloalkyl, —C₀₋₈alkyl-N(C₀₋₈alkyl)-C₀₋₈alkylheterocycloalkyl, —C₀₋₈alkyl-N(C₀₋₈alkyl)-C₀₋₈alkylaryl, —C₀₋₈alkyl-N(C₀₋₈alkyl)-C₀₋₈alkylhetaryl, —C₀₋₈alkyl-N(C₀₋₈alkyl)(C₀₋₈alkyl), C₂₋₈alkenyl, C₂₋₈alkynyl, NO₂, CN, CF₃, OCF₃, or OCHF₂ substituents; wherein said ring in each instance independently optionally includes one or more heteroatoms other than the nitrogen; m is 0, 1, 2, or 3; n is 0, 1, 2, 3, or 4: aa is 0 or 1; and provided that Formula I is not trans-4-[8-amino-1-(7-chloro-4-hydroxy-1H-indol-2-yl)imidazo[1,5-a]pyrazin-3-yl]cyclohexanecarboxylic acid, cis-3-[8-amino-1-(7-chloro-1H-indol-2-yl)imidazo[1,5-a]pyrazin-3-yl]cyclobutanecarboxylic acid, trans-4-{8-amino-1-[7-(3-isopropyl)phenyl-1H-indol-2-yl]imidazo[1,5-a]pyrazin-3-yl}cyclohexanecarboxylic acid or trans-4-{8-amino-1-[7-(2,5-dichloro)phenyl-1H-indol-2-yl]imidazo[1,5-a]pyrazin-3-yl}cyclohexanecarboxylic acid. 46: The method of claim 45, wherein the EGFR kinase inhibitor comprises erlotinib, or a salt thereof. 47: The method of claim 45, additionally comprising administering to said subject one or more other anti-cancer agents. 48: The method of claim 27, wherein the cells of the tumors or tumor metastases are relatively insensitive or refractory to treatment with an EGFR inhibitor as a single agent. 49: The method of claim 42, wherein the cancer is relatively insensitive or refractory to treatment with an EGFR inhibitor as a single agent. 50: The method of claim 45, wherein the cells of the tumors or tumor metastases are relatively insensitive or refractory to treatment with an EGFR inhibitor as a single agent. 51-59. (canceled) 60: The method of claim 27, wherein the patient is in need of treatment for a cancer selected from NSCLC, head and neck squamous cell carcinoma, melanoma, pancreatic, breast and ovarian cancers. 61: The method of claim 60, wherein the EGFR kinase inhibitor comprises erlotinib, or a salt thereof. 62: The method of claim 42, wherein the cancer is selected from NSCLC, head and neck squamous cell carcinoma, melanoma, pancreatic, breast and ovarian cancers. 63: The method of claim 45, wherein the patient is in need of treatment for a cancer selected from NSCLC, head and neck squamous cell carcinoma, melanoma, pancreatic, breast and ovarian cancers. 64-65. (canceled) 66: The method of claim 27, wherein the compound represented by Formula (I) comprises:

67: The method of claim 42, wherein the compound represented by Formula (I) comprises:

68: The method of claim 45, wherein the compound represented by Formula (I) comprises:

69: The method of claim 27, wherein the compound represented by Formula (I) comprises:

70: The method of claim 42, wherein the compound represented by Formula (I) comprises:

71: The method of claim 45, wherein the compound represented by Formula (I) comprises: 